Compositions, methods, and systems for inferring bovine breed

ABSTRACT

Provided herein are methods to discover and use single nucleotide polymorphisms (SNP) for identifying breed, or line and breed, or line composition of a bovine subject. The present invention further provides specific nucleic acid sequences, SNPs, and SNP patterns that can be used for identifying breed or breed combinations for Angus, Holstein, Limousin, Brahman, Hereford, Simmental, Gelbvieh, Charolais and Beefmaster breeds. These patterns can be utilized to manage animals in a feedlot to obtain optimum performance based on known characteristics of specific breeds and identify animals for breeding in selection programs. In another aspect, these patterns can be used to ensure labeling on breed specific branded products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/148,507, filed Apr. 18, 2008, which issued as U.S. Pat. No.8,026,064, which is a continuation of U.S. application Ser. No.10/750,622, filed Dec. 31, 2003, which issued as U.S. Pat. No.7,511,127, which claims the benefit of U.S. Application No. 60/437,482,filed Dec. 31, 2002. The disclosure of each of the above applications isthe entire content of which is incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to genetic markers and more specificallyto polymorphisms associated with bovine breed.

2. Background Information

Classification of individual animals in a livestock population has oftenrelied on a priori groupings of individual animals on the basis ofparentage and registration with a Breed Association. If these criteriaare not known or not available, animals can be classified as a member ofa breed or combination of breeds based on phenotype or geographiclocation. For example, a bovine animal with a black coat color isfrequently classified as an Angus—a breed distinguished by having ablack coat color. Further, a bovine animal with a pronounced hump overthe shoulder region, pendulous ears and loose skin on dewlap and throatis classified as Brahman. These phenotypes such as coat color, ear andhump appearance are readily observable by livestock producers andfrequently used for the basis of breed classification.

Two possible options for classifying an individual bovine animal into apopulation are:

-   -   1) Assign an animal to a population based on known or assumed        parentage, phenotypic appearance or trait value for some        phenotype, or    -   2) From a set of predefined populations (such as breeds), sample        DNA from a number of members of each population to estimate        allele frequencies in each population. Using the allele        frequencies, it is possible to compute the likelihood a given        genotype originated in each population and individuals can be        assigned to population on the basis of these likelihoods        (Pritchard, J. K., et al., Genetics 155: 945-959 (2000)).

Both strategies (above) rely on defining a set of populations. Aclassification based on phenotype or geographic locality may notaccurately describe the genetic structure of a population if similarphenotypes can arise despite differences in genotype (Rosenberg, N. A.,et al., Genetics. 159: 699-713 (2001)). Coat color in cattle isdetermined by one or a few loci that are inherited in a well-knownmanner (Olson, T. A. 1999. Genetics of colour variation. In: Fries, R.and Ruvinsky, A. (eds.) The Genetics of Cattle—CABI Publishing,Wallingford). It is relatively easy for livestock breeders to introgressnew genes for coat color into a population (such as a breed) resultingin phenotypes that were not previously present.

Some beef marketing systems rely on accurate determination of breed ofanimal. Of the 41 brands of beef certified by the USDA, 33 name a breedand of these, 30 name Angus, two name Hereford and one names Piedmontese(Smith, G. C., available on the inter-net atansci.colostate.edu/ran/beef/smith7.pdf). To date, the only methodsavailable to qualify animals for these systems are known or assumedparentage or phenotypic appearance. There is an opportunity to improveaccuracy of individual animal qualification using the allele frequenciesto compute the likelihood that a given genotype originated in specificbreed population.

It is critical to know the breed of cattle entered into and qualifyingfrom branded beef programs when those branded beef programs includebreed specifications. In particular, knowledge of breed composition isimportant for the following:

-   -   1) to verify claims for breed type or breed composition        associated with breed-specific marketing programs. Verification        of claims for breed type or breed composition has not been        possible because no available technology could classify a bovine        animal to a particular population or infer the breed composition        of an individual animal. Currently, the only bovines accepted by        breed are those within the seedstock sector where the records of        individual animals are maintained by Breed Associations or in        commercial populations where cattle are recorded in a        performance registry usually by a Breed Association. These total        breed-identified cattle likely comprise some five percent of the        national beef cattle population. Therefore, standardization of        claims for breed or breed type is very loose and limited to a        small fraction of animals contributing to the beef supply; and    -   2) to implement management strategies for the feeding of cattle        to optimize the pre-harvest growth and development and        post-harvest fabrication of their associated beef products.        Efficiencies in production from feeding, sorting, managing and        marketing cattle can be obtained by identifying and matching        breed-specific phenotypic data for feedlot, carcass and meat        quality traits. Where breeds cannot be distinguished on the        merit of parentage and simply measured phenotypic differences, a        purely genetic analysis provides the most suitable test to        assign animals to a population (Rosenberg, N. A., et al.,        Genetics 159: 699-713 (2001)). Only if a correspondence between        genotype and known parentage classification is established can        these characteristics also serve as appropriate classification        tools.

Accordingly, there remains a need for methods and compositions thatprovide information regarding bovine breed.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of bovinesingle nucleotide polymorphism (SNP) markers that are associated with,and predictive of, bovine breeds including, but not limited to, Angus,Brahman, Charolais, Hereford, Simmental, Gelbvieh, Limousin, andBeefmaster breeds. Accordingly, the present invention provides methodsto discover and use single nucleotide polymorphisms (SNP) foridentifying breed, or line and breed, or line composition of a bovinesubject. The present invention further provides specific nucleic acidsequences, SNPs, and SNP patterns that can be used for identifying breedor breed combinations for Angus, Limousin, Brahman, Hereford, Simmental,Gelbvieh, Charolais and Beefmaster breeds. These patterns can beutilized to manage animals in a feedlot to obtain optimum performancebased on known characteristics of specific breeds. In anotherembodiment, cattle can be marketed prior to harvest based on their breedcharacteristics for meat production. In another aspect, branded productsbased on breed designation can certify their label based on specificbreed markers.

Accordingly, in one embodiment the present invention provides a methodto infer breed of a bovine subject from a nucleic acid sample of thebovine subject, that includes identifying in the nucleic acid sample, atleast one nucleotide occurrence of at least one single nucleotidepolymorphism (SNP) corresponding to position 300 of any one of SEQ IDNOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein the SNP isassociated with a breed, thereby inferring the breed of the bovinesubject. A SNP is associated with a breed when at least one nucleotideoccurrence of the SNP occurs more frequently in subjects of a particularbreed than other breeds in a statistically significant manner, forexample with greater than 80%, 85%, 90%, 95%, or 99% confidence.Therefore, in certain aspects, the methods include identifying whetherthe nucleotide occurrence is a bovine SNP allele identified herein asassociated with bovine breed. In certain aspects, the identified breedincludes, but is not limited to, Angus, Limousin, Brahman, Simmental,Hereford, Gelbvieh or Charolais. Furthermore, in certain aspects, atleast one nucleotide occurrence of at least one SNP listed in Table 5 orTable 3A as associated with Angus cattle is identified.

In another embodiment, the present invention provides a method fordetermining a nucleotide occurrence of a single nucleotide polymorphism(SNP) in a bovine sample, that includes contacting a bovinepolynucleotide in the sample with an oligonucleotide that binds to atarget region of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895, and determining the nucleotide occurrence of a single nucleotidepolymorphism (SNP) corresponding to position 300 of any one of SEQ IDNOS:1 to 4868 or SEQ ID NOS:64887 to 64895. The determination typicallyincludes analyzing binding of the oligonucleotide, or detecting anamplification product generated using the oligonucleotide, therebydetermining the nucleotide occurrence of the SNP.

In another embodiment, the present invention provides an isolatedpolynucleotide that includes a fragment of at least 20 contiguousnucleotides, a polynucleotide at least 90% identical to the fragment of20 contiguous nucleotides, or a complement thereof, wherein the isolatedpolynucleotide includes a nucleotide occurrence of a single nucleotidepolymorphism (SNP) associated with breed, wherein the SNP corresponds toposition 300 of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895.

In yet another embodiment, the present invention provides an isolatedoligonucleotide that includes 10 nucleotides, that selectively binds toa target polynucleotide of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895, wherein a terminal nucleotide of the isolated oligonucleotidebinds to position 299, 300, or 301 of any one of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895.

DETAILED DESCRIPTION OF THE INVENTION

The specification hereby incorporates by reference in their entirety,the files contained on the two compact discs filed herewith. Two copiesof each of the two compact discs are filed herewith. The first compactdisc includes a file called “MMI1150 Table 5.doc,” created Dec. 31,2003, which is 4770 kilobytes in size, and a file called “MMI1150 Table6.doc,” created Dec. 31, 2003, which is 8575 kilobytes in size. TheSecond disc includes a sequence listing which is included in a filecalled “MMI1150 SEQUENCE LISTING.txt,” created Dec. 31, 2003, which is88096 kilobytes in size.

The present invention is based in part on the discovery of singlenucleotide polymorphisms (SNPs) that can be used to infer breed of abovine subject. Accordingly, provided herein is a method for inferringthe breed of a bovine subject from a nucleic acid sample of the bovinesubject, by identifying in the sample, a nucleotide occurrence for atleast one single nucleotide polymorphism (SNP), wherein the nucleotideoccurrence is associated with the breed. In certain aspects, the SNPcorresponds to position 300 of any one of SEQ ID NOS:1 to 4868 or SEQ IDNOS:64887 to 64895.

Using the teachings herein, SNPs associated with any breed of cattle canbe identified. Therefore, methods of the present invention for inferringbreed of a bovine subject, can be used to infer the breed of any bovinesubject. For example, the methods can be used to infer a breedincluding, but not limited to, Angus, Limousin, Brahman, Simmental,Hereford, Holstein, Gelbvieh or Charolais cattle. In certain aspects,the methods are used to infer an Angus from a non-Angus breed.Furthermore, the methods of the present invention can be used to assigna breed or breeds an individual animal with a specific probability.Typically, an identified nucleotide occurrence is compared to multipleknown SNP alleles associated with multiple breeds, for example the breedassociated alleles identified herein in Tables 5 and 3A, to infer abreed for a subject from multiple possible breeds.

The Example provided herein illustrates the use of whole genome shotgunsequencing and genotyping analysis to identify SNPs that can be used toinfer breed of a bovine subject. For this analysis, genomic DNAlibraries were constructed from six breeds of cattle (Angus, Limousin,Brahman, Simmental, Holstein and Charolais). The libraries were amixture of 2.5, and 10 kilobase inserts. Libraries from four of thecattle breeds (Angus, Brahman, Simmental and Limousin) were sequenced,using the whole genome shotgun sequencing method developed by CeleraGenomics (Venter, J. C. et al. (2001) Science 291:1304), to a depth ofcoverage sufficient to generate putative SNPs. The distribution ofsequence fragments obtained from the cattle breeds was 32% Angus, 35%Limousin, 33% Brahman, and less than 1% Simmental.

Allele frequencies within breed were determined using simple countingmethods. Four thousand eight hundred and seventy seven breed specificmarkers (See SEQ ID NOS:1 to 4868 (Tables 5 and 6, included on a compactdisc filed herewith) and SEQ ID NOS:64887 to 64895 (Tables 3A and 3B))were identified by analysis of over 19,000 markers tested. Details ofthe SNP markers that were identified as informative for breed, arelisted in Tables 5 and 6 (filed herewith on a compact disc) and Tables3A and 3B. Tables 3A and 5 provide the names of the identified SNPmarkers, breed specific alleles, their associated breed, allelefrequencies, and breeds for which the alleles, and therefore SNPs, aremost significantly associated (referred to as “major breeds” in thetable). A SNP was identified as being associated with a breed when thefrequency of at least one allele was significantly greater for at leastone breed than for other breeds. Tables 3B and 6 provide primersequences (See “Forward,” “Reverse,” and “Extension”) that were used toamplify a region that includes the SNP, and amplicon sequences thatindicate the nucleotide occurrences for the SNP that were identified inbrackets within the sequence.

Accordingly, in one aspect, the method identifies at least onenucleotide occurrence of at least one SNP listed in Table 5 or Table 3Aas associated with Angus cattle. In another aspect, the methodidentifies at least one nucleotide occurrence of at least one SNP listedin Table 5 or Table 3A as associated with Brahman cattle. In anotheraspect, the method identifies at least one nucleotide occurrence of atleast one SNP listed in Table 5 or Table 3A as associated with Limousincattle. In yet another aspect, the method identifies at least onenucleotide occurrence of at least one SNP listed in Table 5 or Table 3Aas associated with Simmental cattle. In still another aspect, the methodidentifies at least one nucleotide occurrence of at least one SNP listedin Table 5 or Table 3A as associated with Hereford cattle. In anotheraspect, the method identifies at least one nucleotide occurrence of atleast one SNP listed in Table 5 or Table 3A as associated with Charolaiscattle. In another aspect, the method identifies at least one nucleotideoccurrence of at least one SNP listed in Table 5 or Table 3A asassociated with Gelbvieh cattle.

In certain aspects, the method further includes marketing the bovinesubject prior to harvest based on the inferred breed. As indicatedherein, some beef marketing systems rely on accurate determination of abreed of an animal. For example, in certain aspects the inferred breedis used to assign beef of a bovine subject to a USDA certified brandbefore the subject is harvested.

In another aspect, the method further includes managing the bovinesubject in the feedlot to obtain improved performance based on knowncharacteristics of the inferred breed for the subject. In fact, the SNPsand methods of the present invention can be used to identify thepercentage of each breed comprising an individual animal. In thisaspect, for example, animals arriving to the feedlot can have a DNAsample taken at processing. The sample can be screened against breedspecific DNA markers to determine the percentage of breed per animalbased on the breed specific marker panel and the degree of accuracyrequired. These data can be used to manage feedlot cattle for specificgrowth and development traits. In one aspect, cattle identified from theexotic breed type (Charolais, Gelbvieh, Limousin, and Simmental) havehigh growth rate and large harvest sizes. These cattle can be managed tomaximize growth rate and lean meat yield. Cattle identified as Englishtype (Angus and Hereford) produce high quality meat products and can bemarketed into the high quality yield grade.

In other examples, methods of the present invention further includeidentifying a nucleotide sequence of a hypermutable sequence in thesample, and inferring breed based on at least one SNP nucleotideoccurrence and the nucleotide sequence of the hypermutable sequence.Hypermutable sequences include, for example, microsatellite nucleic acidsequences

In another aspect, a method of the present invention further includesbranding or marketing a product of the bovine subject based on theinferred breed, including marketing the product under a trademarkspecific for breed. Breed specified products can certify that theproduct is in fact from the breed labeled, thereby increasing consumerconfidence in a product and increasing the value of a product.

The method can include a determination of the nucleotide occurrence ofat least 2 SNPs. At least 2 SNPs can form all or a portion of ahaplotype, wherein the method identifies a haplotype allele that isassociated with a specific breed. Furthermore, the method can includeidentifying a diploid pair of haplotype alleles.

In another embodiment, the present invention provides a method forsorting one or more bovine subjects, that includes inferring a breed fora first bovine subject from a nucleic acid sample of the first bovinesubject, by identifying a nucleotide occurrence of at least one singlenucleotide polymorphism (SNP) corresponding to position 300 of at leastone of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein theSNP is associated with breed. The first bovine subject is sorted basedon the inferred breed. The method can then be repeated for additionalsubjects, thereby sorting bovine subjects. The bovine subjects can besorted, for example, based on whether they are Angus, Limousin, Brahman,Simmental, Hereford, Gelbvieh or Charolais cattle.

In another embodiment, the present invention provides a method ofproviding labeling accuracy for breed identified meat products, thatincludes inferring breed of a bovine candidate for use in branded meatproducts from a nucleic acid sample of the bovine candidate by a methodcomprising identifying the nucleotide occurrence of at least one singlenucleotide polymorphism (SNP) corresponding to position 300 of at leastone of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein theSNP is associated with breed; and determining whether to brand theharvested product based on the inferred breed. This method providesquality assurance guarantees of the label brand. In certain aspects, forexample, the inferred breed is Angus, Limousin, Brahman, Simmental,Hereford, Gelbvieh or Charolais.

In yet another embodiment, the present invention provides a method forbreeding a bovine subject, that includes inferring breed of a bovinecandidate for use in breeding programs from a nucleic acid sample of thebovine candidate by a method that includes identifying the nucleotideoccurrence of at least one single nucleotide polymorphism (SNP)corresponding to position 300 of at least one of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895, wherein the SNP is associated with breed. Adetermination is made on whether to select the individual for use inbreeding programs based on the inferred breed, thereby breeding thebovine subject.

The selection is implemented, in certain aspects, at the elite orbreeding nucleus level or at the multiplier or foundation animal level.In another embodiment, the present invention provides a bovine subjectresulting from the selection and breeding.

In another embodiment, the present invention provides a method foridentifying a bovine single nucleotide polymorphism (SNP) informative ofbreed, that includes performing whole genome shotgun sequencing of abovine genome, and genotyping at least two bovine subjects from at leasttwo breeds, thereby identifying the bovine single nucleotidepolymorphisms informative of breed. The Example provided herein,illustrates the use of this method to identify breed SNPs.

As used herein, the term “at least one”, when used in reference to agene, SNP, haplotype, or the like, means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,etc., up to and including all of the haplotype alleles, genes,haplotypes, and/or SNPs of the bovine genome. Reference to “at least asecond” gene, SNP, haplotype or the like, means two or more, i.e., 2, 3,4, 5, 6, 7, 8, 9, 10, etc., bovine genes, SNPs, haplotypes, or the like.

Polymorphisms are allelic variants that occur in a population that canbe a single nucleotide difference present at a locus, or can be aninsertion or deletion of one, a few or many consecutive nucleotides. Assuch, a single nucleotide polymorphism (SNP) is characterized by thepresence in a population of one or two, three or four nucleotides (i.e.,adenosine, cytosine, guanosine or thymidine), typically less than allfour nucleotides, at a particular locus in a genome such as the humangenome. It will be recognized that, while the methods of the inventionare exemplified primarily by the detection of SNPs, the disclosedmethods or others known in the art similarly can be used to identifyother types of bovine polymorphisms, which typically involve more thanone nucleotide. A SNP is associated with a breed when at least onenucleotide occurrence of the SNP occurs more frequently in subjects of aparticular breed in a statistically significant manner, for example withgreater than 80%, 85%, 90%, 95%, or 99% confidence. A bovine “SNPallele” is a nucleotide occurrence of a SNP within a population ofbovine animals.

In another embodiment, the present invention provides an isolatedpolynucleotide that includes a fragment of at least 20 contiguousnucleotides of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895, a polynucleotide at least 90% identical to the 20 contiguousnucleotide fragment, or a complement thereof, wherein the isolatedpolynucleotide includes a nucleotide occurrence of a single nucleotidepolymorphism (SNP) that corresponds to position 300 of SEQ ID NOS:1 to4868 or SEQ ID NOS:64887 to 64895.

In certain aspects, the isolated polynucleotide, for example, includes afragment of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200,250, 500, or 600 contiguous nucleotides of any one of SEQ ID NOS:1 to4868 or SEQ ID NOS:64887 to 64895. In another aspect, the isolatedpolynucleotide is at least 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99,or 99.5% identical to SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895,for example. Typically, the isolated nucleotide includes a region thatis contiguous with a region of any one of SEQ ID NOS:1 to 4868 or SEQ IDNOS:64887 to 64895 that includes position 300. In certain aspects, theisolated polynucleotide consists of any one of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895 in its entirety. In other aspects, theisolated polynucleotide consists of any one of SEQ ID NOS:4869 to 19472or SEQ ID NOS:64896 to 64922.

The polynucleotide or an oligonucleotide of the invention can furtherinclude a detectable label. For example, the detectable label can beassociated with the polynucleotide at a position corresponding toposition 300 of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895. As discussed in more detail herein, the labeled polynucleotidecan be generated, for example, during a microsequencing reaction, suchas SNP-IT™ reaction.

Detectable labeling of a polynucleotide or oligonucleotide is well knownin the art. Particular non-limiting examples of detectable labelsinclude chemiluminescent labels, fluorescent labels, radiolabels,enzymes, haptens, or even unique oligonucleotide sequences.

In another embodiment, the present invention provides an isolated vectorthat includes a polynucleotide disclosed hereinabove. The term “vector”refers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of a nucleic acidsequence.

Methods that are well known in the art can be used to construct vectors,including in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination/genetic techniques (See, for example, thetechniques described in Maniatis et al. 1989 Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporatedherein in its entirety by reference).

In another aspect, the present invention provides an isolated cell thatincludes the vector. The cell can be prokaryotic or eukaryotic.Techniques for incorporated vectors into prokaryotic and eukaryoticcells are well known in the art. In certain aspects, the cells arebovine cells. In other aspects, the cells are bacterial cells. In stillother aspects, the cells are human cells.

In another aspect, the present invention provides a primer pair thatbinds to a first target region and a second target region of SEQ IDNOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein the first primer ofthe primer pair and a second primer of the primer pair are at least 10nucleotides in length and bind opposite strands of the target regionlocated within 3000 nucleotides of a position corresponding to position300 of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, andprime polynucleotide synthesis from the target region in oppositedirections across position 300. In another embodiment, provided hereinis a primer pair that binds to a first target region and a second targetregion of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein afirst primer of the primer pair and a second primer of the primer pairare at least 10 nucleotides in length, bind opposite strands of thetarget region, and prime polynucleotide synthesis from the target regionin opposite directions across position 300 of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895.

In another embodiment, the present invention provides an isolatedoligonucleotide that selectively binds to a target polynucleotide thatincludes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 300,500, or 600 nucleotides SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895, or a complement thereof, wherein the terminal nucleotidecorresponds to position 299, 300, or 301. In another embodiment, thepresent invention provides an isolated oligonucleotide that includes 10nucleotides, which selectively binds to a target polynucleotide of anyone of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, wherein aterminal nucleotide of the isolated oligonucleotide binds to position298, 299, 300, 301, or 302 of any one of SEQ ID NOS:1 to 4868 or SEQ IDNOS:64887 to 64895. The oligonucleotide can be, for example, 10, 15, 20,25, 50, or 100 nucleotides in length. In certain aspects, the terminalnucleotide binds to position 300 of any one of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895.

In another embodiment, the present invention provides an isolatedoligonucleotide pair effective for determining a nucleotide occurrenceat a single nucleotide polymorphism (SNP) corresponding to position 300of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, whereineach isolated oligonucleotide comprises at least 5 nucleotides from SEQID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895 and wherein the terminalnucleotide of each oligonucleotide pair is complementary to a differentnucleotide at position 300 of any one of SEQ ID NOS:1 to 4868 or SEQ IDNOS:64887 to 64895 or a complement thereof. In certain aspects, thespecific binding pair member is a substrate for a primer extensionreaction.

As used herein, “about” means within ten percent of a value. Forexample, “about 100” would mean a value between 90 and 110.

The term “haplotypes” as used herein refers to groupings of two or moreSNPs that are physically present on the same chromosome which tend to beinherited together except when recombination occurs. The haplotypeprovides information regarding an allele of the gene, regulatory regionsor other genetic sequences affecting a trait. The linkage disequilibriumand, thus, association of a SNP or a haplotype allele(s) and a bovinebreed can be strong enough to be detected using simple geneticapproaches, or can require more sophisticated statistical approaches tobe identified.

Numerous methods for identifying haplotype alleles in nucleic acidsamples are known in the art. In general, nucleic acid occurrences forthe individual SNPs are determined and then combined to identifyhaplotype alleles. There are several algorithms for haplotypereconstruction based on pedigree analysis. These are the MaximumLikelihood methods ((Excofier, L., and Slatkin, M., Mol. Biol. Evol. 12:921-927 (1995)), the parsimony method created by Clark, A. G., Mol.Biol. Evol. 7: 111-122 (1990) and the phase reconstruction method ofStephens, M., et al., Am. J. Hum. Genet. 68:978-989, 2001, which isincorporated herein by reference). These methods can be applied to thedata generated, regarding individual nucleotide occurrences in SNPmarkers of the subject, in order to determine alleles for each haplotypein a subject's genotype. Alternatively, haplotypes can also bedetermined directly, for each pair of sites, by allele-specific PCR(Clark, A. G. et al., Am. J. Hum. Genet. 63: 595-612 (1998).

As used herein, the term “infer” or “inferring”, when used in referenceto a breed, means drawing a conclusion about a breed using a process ofanalyzing individually or in combination, nucleotide occurrence(s) ofone or more SNP(s), which can be part of one or more haplotypes, in anucleic acid sample of the subject, and comparing the individual orcombination of nucleotide occurrence(s) of the SNP(s) to knownrelationships of nucleotide occurrence(s) of the SNP(s) and the breed.As disclosed herein, the nucleotide occurrence(s) can be identifieddirectly by examining nucleic acid molecules, or indirectly by examininga polypeptide encoded by a particular gene where the polymorphism isassociated with an amino acid change in the encoded polypeptide.

Relationships between nucleotide occurrences of one or more SNPs orhaplotypes and a breed can be identified using known statisticalmethods. A statistical analysis result which shows an association of oneor more SNPs or haplotypes with a breed with at least 80%, 85%, 90%,95%, or 99% confidence, or alternatively a probability of insignificanceless than 0.05, can be used to identify SNPs and haplotypes. Thesestatistical tools may test for significance related to a null hypothesisthat an on-test SNP allele or haplotype allele is not significantlydifferent between groups with different traits. If the significance ofthis difference is low, it suggests the allele is not related to abreed.

In diploid organisms such as bovines, somatic cells, which are diploid,include two alleles for each single-locus haplotype. As such, in somecases, the two alleles of a haplotype are referred to herein as agenotype or as a diploid pair, and the analysis of somatic cells,typically identifies the alleles for each copy of the haplotype. Methodsof the present invention can include identifying a diploid pair ofhaplotype alleles. These alleles can be identical (homozygous) or can bedifferent (heterozygous). Haplotypes that extend over multiple loci onthe same chromosome include up to 2 to the Nth power alleles where N isthe number of loci. It is beneficial to express polymorphisms in termsof multi-locus (i.e. multi SNP) haplotypes because haplotypes offerenhanced statistical power for genetic association studies. Multi-locushaplotypes can be precisely determined from diploid pairs when thediploid pairs include 0 or 1 heterozygous pairs, and N or N−1 homozygouspairs. When multi-locus haplotypes cannot be precisely determined, theycan sometimes be inferred by statistical methods. Methods of theinvention can include identifying multi-locus haplotypes, eitherprecisely determined, or inferred.

A sample useful for practicing a method of the invention can be anybiological sample of a subject, typically a bovine subject, thatcontains nucleic acid molecules, including portions of the genesequences to be examined, or corresponding encoded polypeptides,depending on the particular method. As such, the sample can be a cell,tissue or organ sample, or can be a sample of a biological material suchas blood, milk, semen, saliva, hair, tissue, and the like. A nucleicacid sample useful for practicing a method of the invention can bedeoxyribonucleic (DNA) acid or ribonucleic acids (RNA). The nucleic acidsample generally is a deoxyribonucleic acid sample, particularly genomicDNA or an amplification product thereof. However, where heteronuclearribonucleic acid, which includes unspliced mRNA precursor RNA moleculesand non-coding regulatory molecules such as RNA, is available, a cDNA oramplification product thereof can be used.

Where each of the SNPs of the haplotype is present in a coding region ofa gene(s), the nucleic acid sample can be DNA or RNA, or productsderived therefrom, for example, amplification products. Furthermore,while the methods of the invention generally are exemplified withrespect to a nucleic acid sample, it will be recognized that particularhaplotype alleles can be in coding regions of a gene and can result inpolypeptides containing different amino acids at the positionscorresponding to the SNPs due to non-degenerate codon changes. As such,in another aspect, the methods of the invention can be practiced using asample containing polypeptides of the subject.

In one embodiment, DNA samples are collected and stored in a retrievablebarcode system, either automated or manual, that ties to a database.Collection practices include systems for collecting tissue, hair, mouthcells or blood samples from individual animals at the same time that eartags, electronic identification or other devices are attached orimplanted into the animal. All identities of animals can beautomatically uploaded into a primary database. Tissue collectiondevices can be integrated into the tool used for placing the ear tag.Body fluid samples can be collected and stored on a membrane boundsystem.

The sample is then analyzed on the premises or sent to a laboratorywhere a medium to high-throughput genotyping system is used to analyzethe sample.

The subject of the present invention can be any bovine subject, forexample a bull, a cow, a calf, a steer, or a heifer or any bovine embryoor tissue.

In another aspect, the present invention provides a system fordetermining the nucleotide occurrences in a population of bovine singlenucleotide polymorphisms (SNPs). The system typically includes ahybridization medium and/or substrate that includes at least twooligonucleotides of the present invention, or oligonucleotides used inthe methods of the present invention. The hybridization medium and/orsubstrate are used to determine the nucleotide occurrence of bovine SNPsthat are associated with breed. Accordingly, the oligonucleotides areused to determine the nucleotide occurrence of bovine SNPs that areassociated with a breed. The determination can be made by selectingoligonucleotides that bind at or near a genomic location of each SNP ofthe series of bovine SNPs. The system of the present invention typicallyincludes a reagent handling mechanism that can be used to apply areagent, typically a liquid, to the solid support. The binding of anoligonucleotide of the series of oligonucleotides to a polynucleotideisolated from a genome can be affected by the nucleotide occurrence ofthe SNP. The system can include a mechanism effective for moving a solidsupport and a detection mechanism. The detection method detects bindingor tagging of the oligonucleotides.

Accordingly, in another embodiment, the present invention provides amethod for determining a nucleotide occurrence of a single nucleotidepolymorphism (SNP) in a bovine sample, that includes contacting a bovinepolynucleotide in the sample with an oligonucleotide that binds to atarget region of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895. The nucleotide occurrence of a single nucleotide polymorphism(SNP) corresponding to position 300 of any one of SEQ ID NOS:1 to 4868or SEQ ID NOS:64887 to 64895, is then determined, wherein thedetermination comprises analyzing binding of the oligonucleotide, ordetecting an amplification product generated using the oligonucleotide,thereby determining the nucleotide occurrence of the SNP. In certainaspects, the oligonucleotide binds to a target sequence that includesone of the SNPs, and the nucleotide occurrence is determined based onthe binding of the oligonucleotide to the target sequence.

In another aspect, the bovine polynucleotide is contacted with a pair ofoligonucleotides that constitute a primer pair, and the nucleotideoccurrence is determined using an amplification product generated usingthe primer pair. For example, at least one primer of the primer pair canbe one of SEQ ID NOS:Forward1 to Reverse4868. Furthermore, the primerpair, in certain aspects, is any of the forward and reverse primer pairslisted in Table 6 (Appendix 2).

In certain aspects, the terminal nucleotide of the oligonucleotide bindsto the SNP. In these aspects, the method can include detecting anextension product generated using the oligonucleotide as a primer. Theoligonucleotide, in certain aspects, is any one of SEQ ID NO:4879 to9736 or SEQ ID NO:64914 to 64922.

In other aspects, the terminal nucleotide of each oligonucleotide of apair of oligonucleotides is complementary to a different nucleotide atposition 300 of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to64895, or a complement thereof. In another example, the polynucleotidecan be contacted with a pair of oligonucleotides each comprising adifferent detectable label.

Medium to high-throughput systems for analyzing SNPs, known in the artsuch as the SNPStream® UHT Genotyping System (Beckman/Coulter,Fullerton, Calif.) (Boyce-Jacino and Goelet Patents), the Mass Array™system (Sequenom, San Diego, Calif.) (Storm, N. et al. (2002) Methods inMolecular Biology. 212: 241-262), the BeadArray™ SNP genotyping systemavailable from Illumina (San Diego, Calif.) (Oliphant, A., et al. (June2002) (supplement to Biotechniques), and TaqMan™ (Applied Biosystems,Foster City, Calif.) can be used with the present invention. However,the present invention provides a medium to high-throughput system thatis designed to detect nucleotide occurrences of bovine SNPs, or a seriesof bovine SNPs that can make up a series of haplotypes. Therefore, asindicated above the system includes a solid support or other method towhich a series of oligonucleotides can be associated that are used todetermine a nucleotide occurrence of a SNP for a series of bovine SNPsthat are associated with a trait. The system can further include adetection mechanism for detecting binding of the series ofoligonucleotides to the series of SNPs. Such detection mechanisms areknown in the art.

The system can be a microfluidic device. Numerous microfluidic devicesare known that include solid supports with microchannels (See e.g., U.S.Pat. Nos. 5,304,487, 5,110745, 5,681,484, and 5,593,838).

The SNP detection systems of the present invention are designed todetermine nucleotide occurrences of one SNP or a series of SNPs. Thesystems can determine nucleotide occurrences of an entire genome-widehigh-density SNP map.

Numerous methods are known in the art for determining the nucleotideoccurrence for a particular SNP in a sample. Such methods can utilizeone or more oligonucleotide probes or primers, including, for example,an amplification primer pair that selectively hybridizes to a targetpolynucleotide, which corresponds to one or more bovine SNP positions.Oligonucleotide probes useful in practicing a method of the inventioncan include, for example, an oligonucleotide that is complementary toand spans a portion of the target polynucleotide, including the positionof the SNP, wherein the presence of a specific nucleotide at theposition (i.e., the SNP) is detected by the presence or absence ofselective hybridization of the probe. Such a method can further includecontacting the target polynucleotide and hybridized oligonucleotide withan endonuclease, and detecting the presence or absence of a cleavageproduct of the probe, depending on whether the nucleotide occurrence atthe SNP site is complementary to the corresponding nucleotide of theprobe. These oligonucleotides and probes are another embodiment of thepresent invention.

An oligonucleotide ligation assay (Grossman, P. D. et al. (1994) NucleicAcids Research 22:4527-4534) also can be used to identify a nucleotideoccurrence at a polymorphic position, wherein a pair of probes thatselectively hybridize upstream and adjacent to and downstream andadjacent to the site of the SNP, and wherein one of the probes includesa terminal nucleotide complementary to a nucleotide occurrence of theSNP. Where the terminal nucleotide of the probe is complementary to thenucleotide occurrence, selective hybridization includes the terminalnucleotide such that, in the presence of a ligase, the upstream anddownstream oligonucleotides are ligated. As such, the presence orabsence of a ligation product is indicative of the nucleotide occurrenceat the SNP site. An example of this type of assay is the SNPlex System(Applied Biosystems, Foster City, Calif.).

An oligonucleotide also can be useful as a primer, for example, for aprimer extension reaction, wherein the product (or absence of a product)of the extension reaction is indicative of the nucleotide occurrence. Inaddition, a primer pair useful for amplifying a portion of the targetpolynucleotide including the SNP site can be useful, wherein theamplification product is examined to determine the nucleotide occurrenceat the SNP site. Particularly useful methods include those that arereadily adaptable to a high throughput format, to a multiplex format, orto both. The primer extension or amplification product can be detecteddirectly or indirectly and/or can be sequenced using various methodsknown in the art. Amplification products which span a SNP locus can besequenced using traditional sequence methodologies (e.g., the“dideoxy-mediated chain termination method,” also known as the “SangerMethod” (Sanger, F., et al., J. Molec. Biol. 94:441 (1975); Prober etal. Science 238:336-340 (1987)) and the “chemical degradation method,”“also known as the “Maxam-Gilbert method” (Maxam, A. M., et al.; Proc.Natl. Acad. Sci. (U.S.A.) 74:560 (1977)), both references hereinincorporated by reference) to determine the nucleotide occurrence at theSNP locus.

Methods of the invention can identify nucleotide occurrences at SNPsusing genome-wide sequencing or “microsequencing” methods. Whole-genomesequencing of individuals identifies all SNP genotypes in a singleanalysis. Microsequencing methods determine the identity of only asingle nucleotide at a “predetermined” site. Such methods haveparticular utility in determining the presence and identity ofpolymorphisms in a target polynucleotide. Such microsequencing methods,as well as other methods for determining the nucleotide occurrence at aSNP locus are discussed in Boyce-Jacino, et al., U.S. Pat. No.6,294,336, incorporated herein by reference, and summarized herein.

Microsequencing methods include the Genetic Bit™ Analysis methoddisclosed by Goelet, P. et al. (WO 92/15712, herein incorporated byreference). Additional, primer-guided, nucleotide incorporationprocedures for assaying polymorphic sites in DNA have also beendescribed (Kornher, J. S. et al, Nucleic Acids Res. 17:7779-7784 (1989);Sokolov, B. P., Nucleic Acids Res. 18:3671 (1990); Syvanen, A.-C., etal., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl.Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al, Hum.Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992);Nyren, P. et al., Anal. Biochem. 208:171-175 (1993); and Wallace,WO89/10414). These methods differ from Genetic Bit™ Analysis in thatthey all rely on the incorporation of labeled deoxynucleotides todiscriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A.-C., et al. Amer. J. Hum. Genet. (1993) 52:46-59 Otherformats for microsequencing include Pyrosequencing (Pyrosequencing AB,Uppsala, Sweden, Alderborn et al (2000) Genome Res. 10:1249-1258).

Alternative microsequencing methods have been provided by Mundy, C. R.(U.S. Pat. No. 4,656,127) and Cohen, D. et al (French Patent 2,650,840;PCT Appln. No. WO91/02087), which discuss a solution-based method fordetermining the identity of the nucleotide of a polymorphic site. As inthe Mundy method of U.S. Pat. No. 4,656,127, a primer is employed thatis complementary to allelic sequences immediately 3′- to a polymorphicsite.

In response to the difficulties encountered in employing gelelectrophoresis to analyze sequences, alternative methods formicrosequencing have been developed. Macevicz (U.S. Pat. No. 5,002,867),for example, describes a method for determining nucleic acid sequencevia hybridization with multiple mixtures of oligonucleotide probes. Inaccordance with such method, the sequence of a target polynucleotide isdetermined by permitting the target to sequentially hybridize with setsof probes having an invariant nucleotide at one position, and variantnucleotides at other positions. The Macevicz method determines thenucleotide sequence of the target by hybridizing the target with a setof probes, and then determining the number of sites that at least onemember of the set is capable of hybridizing to the target (i.e., thenumber of “matches”). This procedure is repeated until each member of aset of probes has been tested.

Boyce-Jacino, et al., U.S. Pat. No. 6,294,336 provides a solid phasesequencing method for determining the sequence of nucleic acid molecules(either DNA or RNA) by utilizing a primer that selectively binds apolynucleotide target at a site wherein the SNP is the most 3′nucleotide selectively bound to the target.

The occurrence of a SNP can be determined using denaturing HPLC such asdescribed in Nairz K et al (2002) Proc. Natl. Acad. Sci. (U.S.A.)99:10575-80, and the Transgenomic WAVE® System (Transgenomic, Inc.Omaha, Nebr.).

Oliphant et al. report a method that utilizes BeadArray™ Technology thatcan be used in the methods of the present invention to determine thenucleotide occurrence of a SNP (supplement to Biotechniques, June 2002).Additionally, nucleotide occurrences for SNPs can be determined using aDNAMassARRAY system (SEQUENOM, San Diego, Calif.). This system combinesproprietary SpectroChips™, microfluidics, nanodispensing, biochemistry,and MALDI-TOF MS (matrix-assisted laser desorption ionization time offlight mass spectrometry).

As another example, the nucleotide occurrences of bovine SNPs in asample can be determined using the SNP-IT™ method (Beckman Coulter,Fullerton, Calif.). In general, SNP-IT™ is a 3-step primer extensionreaction. In the first step a target polynucleotide is isolated from asample by hybridization to a capture primer, which provides a firstlevel of specificity. In a second step the capture primer is extendedfrom a terminating nucleotide triphosphate at the target SNP site, whichprovides a second level of specificity. In a third step, the extendednucleotide trisphosphate can be detected using a variety of knownformats, including: direct fluorescence, indirect fluorescence, anindirect colorimetric assay, mass spectrometry, fluorescencepolarization, etc. Reactions can be processed in 384 well format in anautomated format using a SNPstream™ instrument (Beckman Coulter,Fullerton, Calif.). Reactions can also be analyzed by binding to Luminexbiospheres (Luminex Corporation, Austin, Tex., Cai. H. (2000) Genomics66(2):135-43). Other formats for SNP detection include TaqMan™ (AppliedBiosystems, Foster City, Calif.), Rolling circle (Hatch et al (1999)Genet. Anal. 15: 35-40, Qi et al (2001) Nucleic Acids Research Vol. 29e116), fluorescence polarization (Chen, X., et al. (1999) GenomeResearch 9:492-498), SNaPShot (Applied Biosystems, Foster City, Calif.)(Makridakis, N. M. et al. (2001) Biotechniques 31:1374-80),oligo-ligation assay (Grossman, P. D., et al. (1994) Nucleic AcidsResearch 22:4527-4534), locked nucleic acids (LNATM, Link, TechnologiesLTD, Lanarkshire, Scotland, EP patent 1013661, U.S. Pat. No. 6,268,490),Invader Assay (Aclara Biosciences, Wilkinson, D. (1999) The Scientist13:16), padlock probes (Nilsson et al. Science (1994), 265: 2085),Sequence-tagged molecular inversion probes (similar to padlock probes)from ParAllele Bioscience (South San Francisco, Calif.; Hardenbol, P. etal. (2003) Nature Biotechnology 21:673-678), Molecular Beacons (Marras,S. A. et al. (1999 Genet Anal. 14:151-156), the READIT™ SNP GenotypingSystem from Promega (Madison, Wis.) (Rhodes R. B. et al. (2001) MolDiagn. 6:55-61), Dynamic Allele-Specific Hybridization (DASH) (Prince,J. A. et al. (2001) Genome Research 11:152-162), the Qbead™ system(quantum dot encoded microspheres conjugated to allele-specificoligonucleotides) (Xu H. et al. (2003) Nucleic Acids Research 31:e43),Scorpion primers (similar to molecular beacons except unimolecular)(Thelwell, N. et al. (2000) Nucleic Acids Research 28:3752-3761), andMagiprobe (a novel fluorescence quenching-based oligonucleotide probecarrying a fluorophore and an intercalator) (Yamane A. (2002) NucleicAcids Research 30:e97). In addition, Rao, K. V. N. et al. ((2003)Nucleic Acids Research. 31:e66), recently reported a microsphere-basedgenotyping assay that detects SNPs directly from human genomic DNA. Theassay involves a structure-specific cleavage reaction, which generatesfluorescent signal on the surface of microspheres, followed by flowcytometry of the microspheres. With a slightly different twist on theSequenom technology (MALDI), Sauer et al. ((2003) Nucleic Acids Research31:e63) generate charge-tagged DNA (post PCR and primer extension),using a photocleavable linker.

Accordingly, using the methods described above, the bovine haplotypeallele or the nucleotide occurrence of a bovine SNP can be identifiedusing an amplification reaction, a primer extension reaction, or animmunoassay. The bovine haplotype allele or bovine SNP can also beidentified by contacting polynucleotides in the sample orpolynucleotides derived from the sample, with a specific binding pairmember that selectively hybridizes to a polynucleotide region comprisingthe bovine SNP, under conditions wherein the binding pair memberspecifically binds at or near the bovine SNP. The specific binding pairmember can be an antibody or a polynucleotide.

The nucleotide occurrence of a SNP can be identified by othermethodologies as well as those discussed above. For example, theidentification can use microarray technology, which can be performedwith PCR, for example using Affymetrix technologies and GenFlex Tagarrays (See e.g., Fan et al (2000) Genome Res. 10:853-860), or using abovine gene chip containing proprietary SNP oligonucleotides (See e.g.,Chee et al (1996), Science 274:610-614; and Kennedy et al. (2003) NatureBiotech 21:1233-1237) or without PCR, or sequencing methods such as massspectrometry, scanning electron microscopy, or methods in which apolynucleotide flows past a sorting device that can detect the sequenceof the polynucleotide. The occurrence of a SNP can be identified usingelectrochemical detection devices such as the eSensor™ DNA detectionsystem (Motorola, Inc., Yu, C. J. (2001) J. Am Chem. Soc.123:11155-11161). Other formats include melting curve analysis usingfluorescently labeled hybridization probes, or intercalating dyes(Lohmann, S. (2000) Biochemica 4, 23-28, Herrmann, M. (2000) ClinicalChemistry 46: 425).

The SNP detection systems of the present invention typically utilizeselective hybridization. As used herein, the term “selectivehybridization” or “selectively hybridize,” refers to hybridization undermoderately stringent or highly stringent conditions such that anucleotide sequence preferentially associates with a selected nucleotidesequence over unrelated nucleotide sequences to a large enough extent tobe useful in identifying a nucleotide occurrence of a SNP. It will berecognized that some amount of non-specific hybridization isunavoidable, but is acceptable provide that hybridization to a targetnucleotide sequence is sufficiently selective such that it can bedistinguished over the non-specific cross-hybridization, for example, atleast about 2-fold more selective, generally at least about 3-fold moreselective, usually at least about 5-fold more selective, andparticularly at least about 10-fold more selective, as determined, forexample, by an amount of labeled oligonucleotide that binds to targetnucleic acid molecule as compared to a nucleic acid molecule other thanthe target molecule, particularly a substantially similar (i.e.,homologous) nucleic acid molecule other than the target nucleic acidmolecule. Conditions that allow for selective hybridization can bedetermined empirically, or can be estimated based, for example, on therelative GC:AT content of the hybridizing oligonucleotide and thesequence to which it is to hybridize, the length of the hybridizingoligonucleotide, and the number, if any, of mismatches between theoligonucleotide and sequence to which it is to hybridize (see, forexample, Sambrook et al., “Molecular Cloning: A laboratory manual (ColdSpring Harbor Laboratory Press 1989)).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42EC (moderate stringency conditions); and0.1×SSC at about 68EC (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

The term “polynucleotide” is used broadly herein to mean a sequence ofdeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. For convenience, the term “oligonucleotide” is usedherein to refer to a polynucleotide that is used as a primer or a probe.Generally, an oligonucleotide useful as a probe or primer thatselectively hybridizes to a selected nucleotide sequence is at leastabout 15 nucleotides in length, usually at least about 18 nucleotides,and particularly about 21 nucleotides or more in length.

A polynucleotide can be RNA or can be DNA, which can be a gene or aportion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence,or the like, and can be single stranded or double stranded, as well as aDNA/RNA hybrid. In various embodiments, a polynucleotide, including anoligonucleotide (e.g., a probe or a primer) can contain nucleoside ornucleotide analogs, or a backbone bond other than a phosphodiester bond.In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotideor oligonucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucleic Acids Research (1994)22:5220-5234 Jellinek et al., Biochemistry (1995) 34:11363-11372;Pagratis et al., Nature Biotechnol. (1997) 15:68-73, each of which isincorporated herein by reference). Primers and probes can also becomprised of peptide nucleic acids (PNA) (Nielsen P E and Egholm M.(1999) Curr. Issues Mol. Biol. 1:89-104).

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. (1994) 22:977-986, Eckerand Crooke, BioTechnology (1995) 13:351360, each of which isincorporated herein by reference). The incorporation of non-naturallyoccurring nucleotide analogs or bonds linking the nucleotides or analogscan be particularly useful where the polynucleotide is to be exposed toan environment that can contain a nucleolytic activity, including, forexample, a tissue culture medium or upon administration to a livingsubject, since the modified polynucleotides can be less susceptible todegradation.

A polynucleotide or oligonucleotide comprising naturally occurringnucleotides and phosphodiester bonds can be chemically synthesized orcan be produced using recombinant DNA methods, using an appropriatepolynucleotide as a template. In comparison, a polynucleotide oroligonucleotide comprising nucleotide analogs or covalent bonds otherthan phosphodiester bonds generally are chemically synthesized, althoughan enzyme such as T7 polymerase can incorporate certain types ofnucleotide analogs into a polynucleotide and, therefore, can be used toproduce such a polynucleotide recombinantly from an appropriate template(Jellinek et al., supra, 1995). Thus, the term polynucleotide as usedherein includes naturally occurring nucleic acid molecules, which can beisolated from a cell, as well as synthetic molecules, which can beprepared, for example, by methods of chemical synthesis or by enzymaticmethods such as by the polymerase chain reaction (PCR).

In various embodiments for identifying nucleotide occurrences of SNPs,it can be useful to detectably label a polynucleotide oroligonucleotide. Detectable labeling of a polynucleotide oroligonucleotide is well known in the art. Particular non-limitingexamples of detectable labels include chemiluminescent labels,fluorescent labels, radiolabels, enzymes, haptens, or even uniqueoligonucleotide sequences.

A method of the identifying a SNP also can be performed using a specificbinding pair member. As used herein, the term “specific binding pairmember” refers to a molecule that specifically binds or selectivelyhybridizes to another member of a specific binding pair. Specificbinding pair member include, for example, probes, primers,polynucleotides, antibodies, etc. For example, a specific binding pairmember includes a primer or a probe that selectively hybridizes to atarget polynucleotide that includes a SNP loci or that hybridizes to anamplification product generated using the target polynucleotide as atemplate.

As used herein, the term “specific interaction,” or “specifically binds”or the like means that two molecules form a complex that is relativelystable under physiologic conditions. The term is used herein inreference to various interactions, including, for example, theinteraction of an antibody that binds a polynucleotide that includes aSNP site; or the interaction of an antibody that binds a polypeptidethat includes an amino acid that is encoded by a codon that includes aSNP site. According to methods of the invention, an antibody canselectively bind to a polypeptide that includes a particular amino acidencoded by a codon that includes a SNP site. Alternatively, an antibodymay preferentially bind a particular modified nucleotide that isincorporated into a SNP site for only certain nucleotide occurrences atthe SNP site, for example using a primer extension assay.

A specific interaction can be characterized by a dissociation constantof at least about 1×10−6 M, generally at least about 1×10−7 M, usuallyat least about 1×10−8 M, and particularly at least about 1×10−9 M or1×10−10 M or less. A specific interaction generally is stable underphysiological conditions, including, for example, conditions that occurin a living individual such as a human or other vertebrate orinvertebrate, as well as conditions that occur in a cell culture such asused for maintaining mammalian cells or cells from another vertebrateorganism or an invertebrate organism. Methods for determining whethertwo molecules interact specifically are well known and include, forexample, equilibrium dialysis, surface plasmon resonance, and the like.

The invention also relates to kits, which can be used, for example, toperform a method of the invention. Thus, in one embodiment, theinvention provides a kit for identifying nucleotide occurrences orhaplotype alleles of bovine SNPs. Such a kit can contain, for example,an oligonucleotide probe, primer, or primer pair, or combinationsthereof for identifying the nucleotide occurrence of at least one bovinesingle nucleotide polymorphism (SNP) associated with breed, such as aSNP corresponding to position 300 of any one of SEQ ID NOS:1 to 4868 orSEQ ID NOS:64887 to 64895. Such oligonucleotides being useful, forexample, to identify a SNP or haplotype allele as disclosed herein; orcan contain one or more polynucleotides corresponding to a portion of abovine gene containing one or more nucleotide occurrences associatedwith a bovine trait, such polynucleotide being useful, for example, as astandard (control) that can be examined in parallel with a test sample.In addition, a kit of the invention can contain, for example, reagentsfor performing a method of the invention, including, for example, one ormore detectable: labels, which can be used to label a probe or primer orcan be incorporated into a product generated using the probe or primer(e.g., an amplification product); one or more polymerases, which can beuseful for a method that includes a primer extension or amplificationprocedure, or other enzyme or enzymes (e.g., a ligase or anendonuclease), which can be useful for performing an oligonucleotideligation assay or a mismatch cleavage assay; and/or one or more buffersor other reagents that are necessary to or can facilitate performing amethod of the invention. The primers or probes can be included in a kitin a labeled form, for example with a label such as biotin or anantibody. In one embodiment, a kit of the invention provides a pluralityof oligonucleotides of the invention, including one or moreoligonucleotide probes or one or more primers, including forward and/orreverse primers, or a combination of such probes and primers or primerpairs. Such a kit also can contain probes and/or primers thatconveniently allow a method of the invention to be performed in amultiplex format.

The kit can also include instructions for using the probes or primers todetermine a nucleotide occurrence of at least one bovine SNPs.

Population-specific alleles can be used to assign a bovine animal to aparticular breed. These population specific alleles are fixed in thepopulation of interest and absent in comparison populations. The absenceof an allele in a sample of individuals from any one population may bebecause those alleles are truly population-specific or because thefrequency of those alleles is low and the sample obtained from any givenpopulation was small (Taylor, J. F., Patent: PCT/US01/47521). Foradmixed populations, population-specific alleles rarely occur, howeverthe difference in allele frequency between populations may still enabletheir use to infer assignment of individual bovines based to a breed,these are known as population associated alleles (Kumar, P., Heredity91: 43-50 (2003)). Both population specific alleles andpopulation-associated alleles are herein referred to as Breed-SpecificMarkers.

In the present invention, a marker is breed specific if it has adifferent allele frequency in one breed relative to one or more otherbreeds. A similar logic was employed by Kumar, P. (Heredity 91: 43-50(2003)) to genetically distinguish cattle from European Bos taurusbreeds and Indian Bos indicus breeds of cattle.

In the present invention there are 4868 breed-specific SNP markers. Oneor more of these markers could be used to determine breed specificityand/or to assign an individual to one or more breeds with an associatedprobability. These markers could be used alone or in any combination.

There are two broad classes of clustering methods that are used toassign individuals to populations (Pritchard, J. K., et al., Genetics155: 945-959 (2000)). These are: 1) Distance-based methods: Thesecalculate a pairwise distance matrix, whose entries give the distancebetween every pair of individuals. 2) Model-based methods: These proceedby assuming that observations from each cluster are random draws fromsome parametric model. Inference for the parameters corresponding toeach cluster is then done jointly with inference for the clustermembership of each individual, using standard statistical methods. Thepreset disclosure includes the use of all standard statistical methodsincluding maximum likelihood, bootstrapping methodologies, Bayesianmethods and any other statistical methodology that can be employed toanalyze such genome data. These statistical techniques are well known tothose in the art.

Many software programs for molecular population genetics studies havebeen developed, their advantage lies in their pre-programmed complexmathematical techniques and ability to handle large volumes of data.Popular programs used by those in the field include, but are not limitedto: TFPGA, Arlequin, GDA, GENEPOP, GeneStrut, POPGENE (Labate, J. A.,Crop Sci. 40: 1521-1528. (2000)) and Structure. The present disclosureincorporates the use of all of the software disclosed above used toclassify bovines into populations based on DNA polymorphisms as well asother software known in the art.

Structure has been used to determine population structure and inferassignment of individual animals to populations for livestock speciesincluding poultry (Rosenberg, N. A., et al., Genetics. 159: 699-713(2001)) and bovines from South Asia (Kumar, P., Heredity 91: 43-50(2003)).

As used herein, the term “hypermutable” refers to a nucleic acidsequence that is susceptible to instability, this resulting in nucleicacid alterations. Such alterations include the deletion and addition ofnucleotides. The hypermutable sequences of the invention are most oftenmicrosatellite DNA sequences which, by definition, are small tandemrepeat DNA sequences. Thus, a combination of SNP analysis andmicrosatellite analysis may be used to infer a breed of a bovinesubject.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Identification of SNPs that can be Used to Infer Breed

This example illustrates the use of whole genome shotgun sequencing andgenotyping analysis to identify SNPs that can be used to infer breed ofa bovine subject. Genomic DNA libraries were constructed from six breedsof cattle (Angus, Limousin, Brahman, Simmental, Holstein and Charolais).The libraries are a mixture of 2.5 and 10 kilobase inserts. Librariesfrom four of the cattle breeds (Angus, Brahman, Simmental and Limousin)were sequenced using the whole genome shotgun sequencing methoddeveloped by Celera Genomics (Venter, J. C. et al. (2001) Science291:1304), to a depth of coverage sufficient to generate putative SNPs.The distribution of sequence fragments obtained from the cattle breedswas 32% Angus, 35% Limousin, 33% Brahman, and less than 1% Simmental.Upon whole genome assembly of the sequenced fragments, contigs wereformed from consensus sequence, and sequence variants were identifiedand cataloged. 786,777 sequence variants that differed by a singlenucleotide became candidate SNP markers for the high-density SNP map.The relative position of each candidate SNP within the bovine genome wasdetermined using the assembled human genome as scaffolding, creating acandidate map of 242,181 human-mapped markers. Individual markers weretested to determine breed specificity within the cattle population using196 animals representing diverse breeds (Angus, Charolais, Limousin,Hereford, Brahman, Simmental and Gelbvieh) and to ensure Mendeliansegregation (20 trios of Beefmaster parents and progeny). The SNPdetection platform used was the SNP-IT™ system (Beckman Coulter,Fullerton, Calif.). In general, SNP-IT™ is a 3-step primer extensionreaction. In the first step a target polynucleotide is isolated from asample by hybridization to a capture primer, which provides a firstlevel of specificity. In a second step the capture primer is extendedfrom a terminating nucleotide triphosphate at the target SNP site, whichprovides a second level of specificity. In a third step, the extendednucleotide trisphosphate can be detected using a variety of knownformats, including, for example: direct fluorescence, indirectfluorescence, an indirect colorimetric assay, mass spectrometry, andfluorescence polarization. Reactions were processed in an automated 384well format using a SNPstream™ instrument (Beckman Coulter, Fullerton,Calif.).

Allele frequencies within breed were determined using simple countingmethods. Four thousand eight hundred and seventy seven breed specificmarkers (See SEQ ID NOS:1 to 4868 (Tables 5 and 6, filed herewith on acompact disc) and SEQ ID NOS:64887 to 64895 (Tables 3A and 3B)) wereidentified by analysis of over 19,000 markers tested. Details of the SNPmarkers that were identified as informative for breed, are listed inTables 5 and 6 (filed herewith on a compact disc) and Tables 3A and 3B.Tables 3A and 5 provide the names of the identified SNP markers, breedspecific alleles, their associated breed, allele frequencies, and breedsfor which the alleles are most significantly associated (referred to as“major breeds” in the table). Tables 313 and 6 provide primer sequences(See “Forward,” “Reverse,” and “Extension”) that were used to amplify aregion that includes the SNP, and amplicon sequences that indicate thenucleotide occurrences for the SNP that were identified in bracketswithin the sequence.

Table 1 shows the number of SNP markers that had a non-zero allelefrequency in the breed of interest. The fourth column shows the abilityto distinguish a subpopulation from the total population as given byWright's F_(st) statistic. This statistic can take on a value of between0 (meaning no ability to distinguish subpopulations [breeds] from thetotal population) and 1.0 meaning that different breeds can beidentified perfectly. An F_(st) value of zero means that a particularbreed has the same allele frequency as the entire population (Weir, B.S. Genetic Data Analysis II. Methods for Discrete Population GeneticData. Sinauer Associates, Sunderland, Mass. p 174. (1996)). Table 2shows the number of SNP markers that were breed specific.

TABLE 1 Number of SNP markers per breed that have a non-zero allelefrequency and ability to differentrate breeds from the whole population.Values for Wright's F_(st) statistic are the mean of four replicates.Number of Number of SNP markers animals from per breed that haveWright's that breed included a non-zero F_(st) Breed in the analysisallele frequency statistic Angus 27 20 .478 Brahman 27 24 .553 Charolais26 20 .435 Gelbvieh 26 19 .448 Hereford 26 10 .622 Limousin 26 20 .448Simmental 26 20 .411 Beefmaster 20 .343

TABLE 2 Number of Population Specific SNP markers per breed (i.e. atnon-zero allele frequency in one breed and zero allele frequency in allother breeds) Number of Population Breed Specific SNP markers per breedAngus 27 Brahman 2976 Charolais 2 Gelbvieh 3 Hereford 3 Limousin 20Simmental 4

Example 2 Utilizing a Subset of Breed Specific Markers for AssigningAnimals to the Correct Breed Category

For this example, 48 SNP markers were selected from the 4868 markersidentified to evaluate their efficacy for use in a breed specific panel.These 48 markers included MMBT05243, MMBT02545, MMBT14829, MMBT11932,MMBT23373, MBT08423, MMBT19771, MMBT10324, MMBT01611, MMBT08985,MMBT02110, MMBT17611, MMBT09623, MBT06416, MMBT04048, MMBT08023,MMBT11652, MMBT21228, MMBT12147, MMBT00059, MMBT21673, MBT09985,MMBT21682, MMBT06242, MMBT10530, MMBT06978, MMBT21426, MMBT21425,MMBT03692, MMBT11974, MMBT01651, MMBT01127, MMBT08438, MMBT03603,MMBT11644, MMBT14000, MMBT12659, MMBT21705, MMBT05684, MMBT21233,MMBT09242, MMBT09142, MMBT00761, MMBT02076, MMBT05710, MMBT21665,MMBT04130, and MMBT18672 (See Tables 3A and 3B below and Tables 5 and 6(provided on a compact disc filed herewith)). Animals were assigned to apopulation based on their genetic profile of these 48 SNP markers usingthe software structure (Pritchard, J. K., et al., Genetics 155: 945-959(2000)). One hundred and ninety six (196) animals were genotyped in thestudy from six Bos taurus breeds (Angus, Hereford, Simmental, Charolais,Limousin and Gelbvieh) and one Bos indicus (American Brahman) breed ofcattle. One further breed (Beefmaster, an admixture of Brahman, Herefordand Shorthorn cattle) was included in the study—but Breed SpecificMarkers were not chosen specifically for this breed.

TABLE 3A Allele Frequencies Breed Specific Major Marker Allele BreedsAF1 AF2 AF3 AF4 Breeds MMBT19771 C Angus Gelbvieh Hereford 0.59 0.060.75 Angus, Hereford MMBT08985 A Angus Charolais Gelbvieh 0.74 0.05 0.040.2 Angus, Simmental Simmental MMBT21228 T Brahman Charolais 0.55 0.30.03 0.02 Brahman, Charolais Gelbvieh Limousin MMBT12147 C BrahmanCharolais 0.72 0.4 0.34 0.07 Brahman Charolais Gelbvieh SimmentalGelbvieh MMBT00059 G Brahman Charolais 0.82 0.28 0.04 0.19 BrahmanCharolais Limousin Simmental Gelbvieh MMBT03692 G Angus HerefordSimmental 0.11 0.38 0.07 Hereford Angus MMBT01127 C Angus BrahmanCharolais 0.05 0.61 0.05 0.57 Brahman Hereford Hereford MMBT14000 GLimousin Simmental 0.24 0.11 Limousin MMBT21665 T Brahman Charolais 0.430.04 0.1 0.25 Brahman Limousin Simmental Simmental

TABLE 3B Additional bovine SNPs associated with breed SEQ SEQ SEQ SEQ IDID ID ID NOS: NOS: NOS: NOS: 64896- 64905- 64914- 64887- Markers Forward64904 Reverse 64913 Extension 64922 Sequence 64895 MMBT19771 TAAATGGA64896 AGAACCAC 64905 GGATGGCGTTC 64914 TCTAATTGTGTAATTTTCTGCAACTACTCT64887 CCTTTGCT TTGGGGGT CGTCCTATTTA GGGACAAAAATCTTGGTAATTGGATTTTTTAAGTTTTG GG CCCCACTCTCT TAAACKAACAAGTTGTCACAATTGGATAGT GCTGTTGTCATATCTTAAACTATTTGTGCTTATGCTTCAGA G AATTTTCAGTGGATGTACAGTGTATTTTAATGTTTTGCATCAGCTTGTGGTCTCAAATTT GATTTAACTTTGAATTTTAAAATAGATTGACAACTCTGATTATACATTGTCTTCACTAAA TGGACCTTTGCTAAGTTTTGAGTGTRTTCATCCCTACCCCACTCTCTGCTGTTGTCATG [C/G]GAGCTAAAATCCTGGCGGATTCAGCTCCACCCCCAAGTGGTTCTGTCTCCCAGAG TGCTTTGCTGTTACTATGGAAATGCCTGCTTATTAATCTTGCTGTCCTTTTACTACCAAA GCACCTTTAACYGTCTCAAGCAATCAAGTGACGTTACCTACAGCAGGCAAAGGAAGAAGA AGACTGCTTTCGCATCCTACAACTGCAATGCTGATTTTAATCAAGATTTAATCAAGGAGC AGAAGGAACTAAAGCTACTCCAACAGCTCTTTAAAAATATACAAAGCTGGATCTTTCTGA TGCAT MMBT08985 TTCTACTT 64897 AAAAAAGT64906 ACGCACGTCCA 64915 CTCTGCACCCTGACTTTCCTGTCAAGTAGT 64888 CCCCCCTCCTTTAAAA CGGTGATTTGA TTATAATAACAGCTAACATTTATGGAGTGT TAATC AAGATACGAAAGAAACCCT TTGCAATGCACTGGGCTTCCATGCCAAGCG GG TAGGTTTGGGGCTTTACACACCTTGTCTTAATTAATCCTCC C CAGCTACCCCCCAAGGTGTACATATCATTACCTCCACTCTCCTAGTGGAAACTGAGGCTC AGAGAAGCTAAGATCACCCCCCCTAGAAAGTGGCAGGGCCAGGATATGAAAACTGCGCTT AACCACCATGCTCTTCTACTTCCCCCCTCTAATCTGAAAAGAAACCCTTAGGTTTGGGGC [A/T]TCTTTGTTTATAGACTGAAGCCCCGTATCTTTTTTAAAGACTTTTTTTGATGTGG ATCATTTTTAAAGTCTTGATTGAATATGTTACAATATTGTTTCTGTTTAATGTTTTGGTT TTTTGGCCGTGAGACATGTTGGATGTCAGCTCCCCAGCGAGGGATCGAACCTGCACCCCC TGCAGGGTGAAGTCTCAGCCGCTGGACCACCAGGGAAGCCCCTGAAGGCCCCTATTGTAA TGTGTATACTGTCCCCATGACTTAGATGCTTTGCAAGTTCAGATTTTTTATATTCTT TT GTTCAG MMBT21228 ATGGGGTC 64898 ACACACCA64907 AGATAGAGTCG 64916 CTTCTTTCCCCCATTCCACGTGTAGGCACA 64889 ATTCACTTAATCAGAG ATGCCAGCTTT GAGGCCTTTAAGCAGCACCACTTGTACCTG GACTC GTGAACAGGCCTGTGA AGAAGCCCCAAGAAGCTGAGCTGGCAGGAG TAGCTCCACCATCTGGTGCTAAAAAAAGTCC TGAGGTTCATAACCACCATCCGGTTCTTGGCCTTTACATAGCCACCTCTCGCTGAGGTCA GGGACCACTGAGCAGCGGCTGCTACCAGAAAACCACAGCCATTTCTCCCCATGGGGTCAT TCACTTGACTCATGTGCTAATCATCTGCTCTGcATTCAGGCCTGTGACCACAGGCTCTAT GAAGCGGGGGTGCAGCGGGGGAGGGGGGCTGTATGATTTGCGAGGGCTSTGCCATTCTGC TTCTCCCTGTGGAGGTGGTCTGTACCTAAAGGATGACCTCCAGAAAAAGCCCTGACACAA CATGTATTTCCCTTCCCTTCTGAAATTGCTGACTTAAGACTCTTCCTCTTTGGCAAAGGT GCCAGGTAGCAGGCTCAGAGGCACAGATTC MMBT12147ACTGCACA 64899 TCAGTACC 64908 AGGGTCTCTAC 64917CATACATGCATGTGTGTGCAGTCACTTCAG 64890 TAGCGGCC AGTGACTG GCTGACGATCTTCATATCTGACTCTGTAATCCCATGGACTG AT TAATCATG AATTACTATTATAGCCCACCAGTCTCCTCTGTCTACGAGAT A CTTAATAAAAATTCCCAAGCAAGAATACTGGAGTGGGTTGC A CATTTCCTCCTCCAGGAGATCTTCCCRACCTAGGGATTGAACTCACATCTCTTACAGCTC CTGCATTGGCAGACAGATACTTTACCACTGAGCCACCTGGGAAGCCCCCTACATTCCATA CATATTTGGTTCAAACTGCACATAGCGGCCATTTCTAATTACTATTACTTAATAAAAAA [C/T]ACTTTTATAATTATGCATTAACTTCCCAATCATGATTACAGTCACTGGTACTGAG CAAGAACATTAAAAATCCAAGACTAGGCCCTGGAGTCCTGATTAGAATTGTACAGATAAA CTTACAGGTTGGAGAGCAGACACAAGCCAAGTCGTGTCCATATTTTAATGGAAGTTTGCA GTAATCCATGCGAGAATGACCAATATTTCTCAAGTTCTTCTGGAACTAATGGTCTATTGC ATTTTGTATTTAGAGTTATCCTAAATCATACATTTGGGGTATGCCAGTGCAAA TATAAA ATGT MMBT00059 TTCTCCCC 64900 ATTTGATT64909 AGGGTCTCTAC 64918 TCCCTACCCCAGTACCTCAGAATGTGACTA 64891 CACAGAACAGCTCACC GCTGACGATAG TACTTGGCCACAGGGTCTTTAAAGAGGTAA CT TGTCAACTTGGCCTCAGA CAGGGTAAAATAAGGTCATTAAGGTGAGCT AACCTCAAGGACTAAGCCAAAACCTCTAAAGCGCTTATAAG A AAGAGATGAGAACACAACCCACAGGTAACATACAGGGAGGAGGCCGCCTACGGCCAAGGA AAGACGTCTCCATCAAAGGAGATAAGGTTCGTAGGTCGGGAACACACACATGCTACTCTT CTCTGCTCCTGTGGTTCTCCCCCACAGAACCTTAAGTTGGCCTCAGAAACCCTCAAGGAA [A/G]TACTTCAAGCTGCCACTATCAACCAACCAGGGTTGACAGGTGAGCTAATCAAATG ACAGGAGAGCTTCCTGCTTCGATGCCCGGATGACCCACTGGGCTTCACTAAACCCCAGCT CCATGTGGACCCACTCCTTCCTGAGGCCTCCGTCTCCCCGTCCGTGAGACGCTGTCTACA GCCCCTGCACGTACCTCCGTGAACTTGTCGGCCACCATGTAACGGACGCGCCAGGACTTG TCCTCAGCAGCCTGGCGCAGGGTGGGCATCACCAGGGCCTCCAGGTCCTCCTGGGGC AG GAGCTG MMBT03692 ACAAGAAG 64901 ATCTTGAA64910 CGTGCCGCTCG 64919 CTTCTCATGCAAGTAAGATAAACAGAAAAT 64892 TAAAGGTGGGAATAGT TGATAGAATAA ATTGTCTGAAGTTACTAATCCAGGTTTCAT ATTGTTAG TACTTTAATTCTCTTATCT TGAATACCTTGTTTAGGTAGAGCAGAAAGT ACTA ACACTT ATAAAAAGGATATGAAGAAAAAAAGACAAGTGTAATGGTCA T TACTCAATTCTAGTGTTTCTAATGACTGAGTCAAATATGTAACAATTTATTTGTGTGTTT GTAATATTTTTAAAAGTTCATTCTGATGACCTTTCAATATTCTTAAATTCTCTTATCTAT AAAAAGGATAAGTAAAGGTGATTGTTAGACTAATAATTCTCTTATCTATAAAAAGGATT [A/G]TTTCCATGAGTTCATTCTCATCTTTTAAGAAATGGAATATATTTTGAAGTGTTTA AAGTAACTATTCCTTCAAGATCACCCAAATCTACTCCTAATCACAATTTAATTAATTTTA GCTAACACATGACTGGAGGTGTAGGTACTTAGATTATTCCAAGTCTCCACAGTCAGCCAT GTTTTCCAGTGGCTGCTGATCATGACATCAGGCCCTGGTAAGACCTCCATTTTGGCCATT TTCATTATTGATGTAGGTTACATCAGACTAACAGTGGCTGAATATCAGCTGTTAGGCT G GCGAAGA MMBT01127 TAGCTCTG 64902 TAAGTGCT64911 GCGGTAGGTTC 64920 GGGTTAAATGAGAGATTGGAGACAGCTTTG 64893 TAAAAAGAGCTCTACC CCGACATATCC TAGCCTATTGAAACAAAGTATGCCCTCAAC ATATTATA ACATGAGTAACTATTA AAACAGTTACTGATTGATTGGTCAAGTCTA ATGCTAAC GTTGAAGAATATCAGACACTGGATACATTTAAAGCACATGA C TAAAAAATGTACAGTTTTCTGAATTTGTTATAAGTTCTGCTGSAATCTGTCACATTGATA AATTGGTCATAAATGACTATTATCTGTGAACATTTGTATGAATTCACCATGTTTTTTATT TAGCTCTGTAAAAAGAATATTATAATGCTAACTGCCAGTAACTATTAGTTGAAGAATAC [A/C]TCCTTGAGCAGTTCAGTGCTTGGCATGTGGTAGAGCAGCACTTAGTAAGTGTTTG TTGAGTCAAAGAACAAATTATTAACAACTTCTAATCAAGGCCTTTGATTCTTCAGCAATT AAATCTCTTTATTCTCATTTTTTACTTTATGGAGAAATGCTGACCAATATGATTTAAGTC ACATACATAATTTTATGTTTTTCAGTATCCACGTTTGAAAAAGTAAAAAGAAACAGCTGA AATTAATTTTAATAATGTATTTTATTTAASCCATGTCAATATATTATGGTTTTG ATATA TAATC MMBT14000 ACCGATGC 64903 TTCTTTCG64912 GGATGGCGTTC 64921 AACTGTCCTGGGTTAGGGAATCCCTGAGCA 64894 TCACTGCTTCCGGGGT CGTCCTATTGT ATAGTCAATTAGGAACAGAGCGCTCGCCTC CA CG GCCCCGTTTAATAAGTTTAGTACTGAGTCATCTGCAGACCC AGCGGAGGAYTTGCTAAAGACGTGAAAAGATAAAGAGGGTA G GGAGAGGAAGGAGGGAGGGAGAGGAAGGGAGAGACAGAGGGAAACAGGGACAGACACAGA GAAATATCCCTGGTCACCATCAACAGTTACGGAACTTCCCACAGTCCCACTCCCTCCACC GATGCTCACTG[A/G]GGCTTGCAGAGGTGCCCCGGATCAGCCCGGCCGCCGCCAGCGCC CCKCAAGRGGGGGCCCGGATACGCCCGCGACCCCGGACGAAAGAAGCCTGCCCGCCCCCC GGGCCCGCACACCTCCTCCTTCCGCGAGCGCTTGGACACTTTCTTCTCCATCTTAGCGGC CGTCTTCTCCGCCCCGCGGCCCTTCTTCTCTTTCTTGCCCTTTTTGCCCATCGCGCCGGA TCCAAGGAGGCTTCTGARCCGCCTGTGAGACTCTAGCGGCGCGCCGCGCGCGGCGCGCAG GAAATACGTGCTgGGG MMBT21665 TGTGGCAA 64904CATGTATG 64913 AGAGCGAGTGA 64922 TCTTTTTGCATTTATTCTTTTCTTCCCTTT 64895ATGGCATA CGGATGGA CGCATACTATT TAAAAAATAACTTTATTTATTTACTTATTT CG TGTAGGCTTACTGT TTGTCTGCTAGGTCTGCATTGCTGCTCAGG GGTGATGAGACCTTTTCCCCAGTTGTGGCAAGTGAGGCCTG A TTCTCTGGTTTCAGGGTGGGCTTCTCACTGCCGTGGCTTCTCACTGCAGTGGCTTCTCGT GTGGAGTATGGGCCGTAGGGCACATGGGCTTTGGTAGTTACACCCTGGGGCTCTAGAGCA CAYGCTCAATATTTGTGGCAAATGGCATCACCACAGTAAGCCTAACATCCATCCGCATAA ACCC[A/G]TGTCTCATCACCACAGTAAGCCTAACATCCATCCGCATACATGGTTGCAAT YATTTTTTCCTAGTGATGAGAACTTTTAAGATCTATTCTCTTGGCACGGCATTCTTAAAG TGCTTATTTTTTCCACTTAAAAAAAATCCAAGTTGGAAGCAGCATGTTATATTAAGAGAT TCTGCATGTAATATGGGCATCTTAAATTTACCATGAAACTGAATTTTTTGCCTTAGAGAT GCAGAAGCTAGAATAATGGATATTATAATTCATGCTCAGTGATGCTTCAGACAACAAATG TGACCTGTT

The number of populations K in the input file was eight. The value of Kwas chosen by sampling 100 SNP markers at random from the population andimputing a value of K from 4 to 12. The statistic ln Pr(X|K) is thelikelihood of the data given the assumed number of populations, where Xis the genotypes of the sampled individuals and K is number of inferredpopulations (Rosenberg, N. A., et al., Genetics. 159: 699-713 (2001)).Three values of K, (K=7 to 9) gave the greatest likelihood of being thetrue number of populations in the study. The value of K=8 was chosen forfuture studies—the same number of known breeds in the population. Thesimulation was run with a burn in period of 25,000 iterations and aruntime of 100,000 iterations for four replicates. Input parametersincluded in the model are shown in Table 4.

TABLE 4 Parameters used in the model Number n (number B (number K(number Burn in Run of SNP of of breeds of inferred period periodmarkers animals) in input) populations) (iterations) (iterations) 48 1968 8 20,000 100,000

The structure program uses a model-based clustering method that employsa Markov Chain to estimate the posterior distribution (q) of eachindividual's admixture coefficient. The mean of this posteriordistribution ({circumflex over (q)}_(k) ^((i))) represents the mean ofthe proportion of an animal i's genome that is derived from a parentalpopulation (k) where Σ_(k)q_(k)=1, i=1, . . . 196 animals and k=1, . . .7 breeds. The higher this value of {circumflex over (q)}, the greaterthe likelihood an animal is derived from a particular parentalpopulation k.

Table 7 shows the mean probability of assignment to a cluster for eachof the eight breeds in the dataset. On average, animals from each breedwere assigned to their own clusters with high probability for all of thepure-breeds represented (probability of cluster assignment ranged from0.802 for Charolais to 0.949 for Brahman). The composite breed,Beefmaster had a lower probability of assignment to its own cluster(0.539) than the seven pure breeds but a significantly non-zero (0.269)probability of assignment to the Brahman cluster—one of the parentalbreeds of Beefmaster. The results show that using genotype informationalone, animals from the same breed were assigned to their respectivepopulation clusters with a high probability. Table 8 shows the meanindividual admixture coefficient ({circumflex over (q)}) per animal asan estimate of probability each individual animal belonging to aparticular cluster with K=8 different clusters.

TABLE 7 Probability of cluster assignment with 48 Breed-Specific SNPmarkers. Cluster Breed 1 2 3 4 5 6 7 8 Angus .009 .002 .937 .007 .005.008 .008 .023 Brahman .005 .949 .020 .004 .003 .006 .006 .008 Charolais.022 .016 .010 .064 .012 .802 .032 .042 Gelbvieh .011 .003 .024 .067.022 .023 .843 .007 Hereford .948 .002 .004 .008 .004 .007 .022 .005Limousin .023 .002 .004 .017 .922 .013 .012 .006 Simmental .008 .003.006 .924 .024 .013 .016 .006 Beefmaster .027 .269 .022 .039 .009 .051.043 .539 Results are the mean of four replicate runs of structure.

TABLE 8 Mean individual admixture coefficient ({circumflex over (q)})per animal as an estimate of probability each individual animalbelonging to a particular cluster with K = 8 different clusters. Breedof Inferred population animal 1 2 3 4 5 6 7 8 Angus 0.01 0.01 0.00 0.000.01 0.00 0.01 0.97 Angus 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.99 Angus0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.96 Angus 0.00 0.00 0.00 0.00 0.000.00 0.01 0.97 Angus 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.99 Angus 0.010.00 0.00 0.00 0.01 0.00 0.00 0.98 Angus 0.01 0.01 0.01 0.00 0.00 0.000.00 0.96 Angus 0.18 0.01 0.02 0.00 0.13 0.02 0.05 0.59 Angus 0.01 0.020.01 0.00 0.01 0.00 0.00 0.95 Angus 0.02 0.01 0.01 0.00 0.05 0.01 0.020.89 Angus 0.01 0.00 0.01 0.00 0.01 0.00 0.02 0.95 Angus 0.01 0.00 0.000.00 0.01 0.01 0.00 0.97 Angus 0.02 0.01 0.01 0.00 0.01 0.01 0.01 0.93Angus 0.00 0.03 0.00 0.01 0.00 0.00 0.00 0.95 Angus 0.00 0.01 0.00 0.000.00 0.00 0.00 0.98 Angus 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.96 Angus0.01 0.01 0.00 0.00 0.05 0.00 0.01 0.91 Angus 0.01 0.01 0.01 0.00 0.000.00 0.00 0.97 Angus 0.09 0.01 0.01 0.00 0.07 0.02 0.05 0.75 Angus 0.010.01 0.00 0.00 0.01 0.01 0.01 0.95 Angus 0.01 0.00 0.00 0.00 0.01 0.010.00 0.97 Angus 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.97 Angus 0.00 0.000.00 0.00 0.00 0.00 0.00 0.98 Beefmaster 0.02 0.01 0.24 0.42 0.16 0.010.13 0.03 Beefmaster 0.01 0.01 0.93 0.02 0.01 0.02 0.00 0.01 Beefmaster0.01 0.02 0.91 0.06 0.01 0.00 0.00 0.00 Beefmaster 0.08 0.14 0.39 0.270.10 0.01 0.01 0.01 Beefmaster 0.03 0.24 0.06 0.63 0.02 0.01 0.01 0.00Beefmaster 0.02 0.01 0.35 0.47 0.01 0.01 0.01 0.13 Beefmaster 0.24 0.110.14 0.45 0.02 0.01 0.03 0.01 Beefmaster 0.03 0.01 0.94 0.01 0.00 0.000.01 0.00 Beefmaster 0.07 0.01 0.81 0.02 0.04 0.01 0.01 0.03 Beefmaster0.08 0.14 0.05 0.65 0.03 0.01 0.03 0.01 Beefmaster 0.04 0.01 0.06 0.670.05 0.01 0.14 0.02 Beefmaster 0.16 0.01 0.18 0.44 0.11 0.01 0.08 0.02Beefmaster 0.01 0.00 0.98 0.00 0.00 0.00 0.00 0.00 Beefmaster 0.01 0.000.96 0.02 0.00 0.00 0.00 0.00 Beefmaster 0.36 0.01 0.59 0.01 0.02 0.010.01 0.00 Beefmaster 0.10 0.12 0.01 0.56 0.13 0.02 0.06 0.01 Beefmaster0.01 0.00 0.96 0.00 0.01 0.00 0.01 0.01 Beefmaster 0.20 0.06 0.06 0.520.09 0.03 0.03 0.01 Beefmaster 0.17 0.05 0.24 0.43 0.03 0.01 0.02 0.05Beefmaster 0.00 0.00 0.98 0.01 0.00 0.00 0.00 0.00 Brahman 0.00 0.000.02 0.96 0.01 0.00 0.00 0.00 Brahman 0.01 0.00 0.01 0.97 0.00 0.00 0.000.00 Brahman 0.01 0.00 0.02 0.94 0.01 0.01 0.01 0.01 Brahman 0.01 0.040.01 0.92 0.01 0.01 0.01 0.00 Brahman 0.01 0.01 0.01 0.92 0.01 0.02 0.020.01 Brahman 0.01 0.01 0.01 0.97 0.01 0.00 0.01 0.00 Brahman 0.00 0.000.00 0.99 0.00 0.00 0.00 0.00 Brahman 0.01 0.01 0.00 0.96 0.01 0.01 0.010.00 Brahman 0.00 0.00 0.00 0.98 0.00 0.00 0.00 0.00 Brahman 0.05 0.000.03 0.85 0.02 0.01 0.01 0.04 Brahman 0.00 0.00 0.00 0.98 0.00 0.00 0.000.00 Brahman 0.00 0.00 0.00 0.98 0.00 0.00 0.00 0.00 Brahman 0.01 0.010.02 0.94 0.01 0.01 0.01 0.00 Brahman 0.00 0.00 0.01 0.98 0.00 0.00 0.000.00 Brahman 0.00 0.00 0.01 0.98 0.00 0.00 0.00 0.00 Brahman 0.00 0.000.01 0.98 0.00 0.00 0.00 0.00 Brahman 0.00 0.00 0.00 0.99 0.00 0.00 0.000.00 Brahman 0.00 0.00 0.01 0.97 0.00 0.00 0.00 0.01 Brahman 0.00 0.000.01 0.97 0.01 0.00 0.00 0.00 Brahman 0.00 0.01 0.00 0.98 0.00 0.00 0.000.00 Brahman 0.00 0.00 0.00 0.99 0.00 0.00 0.00 0.00 Brahman 0.00 0.000.00 0.98 0.00 0.00 0.00 0.00 Brahman 0.00 0.00 0.00 0.98 0.00 0.00 0.000.00 Brahman 0.00 0.00 0.00 0.99 0.00 0.00 0.00 0.00 Brahman 0.00 0.000.00 0.99 0.00 0.00 0.00 0.00 Brahman 0.02 0.00 0.03 0.89 0.02 0.01 0.020.01 Charolais 0.84 0.01 0.02 0.02 0.03 0.01 0.06 0.03 Charolais 0.380.01 0.15 0.01 0.13 0.00 0.13 0.19 Charolais 0.03 0.02 0.89 0.00 0.020.01 0.02 0.01 Charolais 0.72 0.00 0.01 0.01 0.24 0.01 0.01 0.00Charolais 0.74 0.01 0.16 0.01 0.02 0.04 0.01 0.01 Charolais 0.97 0.000.01 0.00 0.01 0.00 0.00 0.00 Charolais 0.96 0.01 0.01 0.00 0.01 0.010.01 0.00 Charolais 0.96 0.00 0.01 0.00 0.02 0.00 0.01 0.00 Charolais0.97 0.00 0.01 0.00 0.01 0.01 0.01 0.00 Charolais 0.92 0.04 0.02 0.020.01 0.00 0.00 0.00 Charolais 0.88 0.00 0.01 0.00 0.01 0.01 0.08 0.00Charolais 0.67 0.02 0.12 0.00 0.15 0.01 0.02 0.02 Charolais 0.96 0.000.01 0.00 0.01 0.01 0.01 0.00 Charolais 0.97 0.00 0.01 0.00 0.01 0.010.01 0.00 Charolais 0.68 0.01 0.02 0.00 0.03 0.01 0.25 0.00 Charolais0.96 0.01 0.01 0.00 0.01 0.01 0.01 0.00 Charolais 0.14 0.01 0.01 0.000.83 0.01 0.01 0.00 Charolais 0.96 0.00 0.00 0.00 0.01 0.01 0.02 0.00Charolais 0.97 0.00 0.00 0.00 0.01 0.00 0.01 0.00 Charolais 0.95 0.010.00 0.01 0.01 0.01 0.01 0.00 Charolais 0.95 0.01 0.01 0.00 0.01 0.000.01 0.00 Charolais 0.96 0.01 0.01 0.00 0.01 0.00 0.01 0.00 Charolais0.94 0.01 0.01 0.00 0.02 0.01 0.02 0.00 Charolais 0.90 0.05 0.01 0.000.01 0.01 0.01 0.00 Charolais 0.96 0.01 0.01 0.00 0.01 0.01 0.01 0.00Charolais 0.95 0.00 0.01 0.00 0.03 0.01 0.01 0.00 Gelbvieh 0.05 0.010.01 0.00 0.93 0.00 0.01 0.00 Gelbvieh 0.01 0.00 0.01 0.05 0.77 0.010.13 0.03 Gelbvieh 0.01 0.00 0.00 0.00 0.96 0.01 0.01 0.00 Gelbvieh 0.010.01 0.00 0.00 0.97 0.00 0.01 0.00 Gelbvieh 0.01 0.01 0.00 0.00 0.960.01 0.01 0.00 Gelbvieh 0.01 0.00 0.00 0.00 0.95 0.03 0.01 0.00 Gelbvieh0.01 0.01 0.00 0.00 0.95 0.01 0.02 0.00 Gelbvieh 0.05 0.06 0.09 0.000.64 0.01 0.01 0.14 Gelbvieh 0.15 0.01 0.01 0.00 0.79 0.01 0.03 0.01Gelbvieh 0.01 0.01 0.00 0.00 0.96 0.00 0.01 0.00 Gelbvieh 0.06 0.01 0.010.00 0.86 0.01 0.04 0.01 Gelbvieh 0.01 0.00 0.01 0.00 0.66 0.01 0.020.29 Gelbvieh 0.01 0.00 0.00 0.00 0.76 0.17 0.05 0.00 Gelbvieh 0.01 0.010.01 0.00 0.51 0.01 0.46 0.00 Gelbvieh 0.01 0.01 0.00 0.00 0.97 0.000.01 0.00 Gelbvieh 0.00 0.00 0.00 0.00 0.97 0.01 0.00 0.00 Gelbvieh 0.010.00 0.00 0.00 0.96 0.01 0.01 0.00 Gelbvieh 0.01 0.02 0.01 0.00 0.320.02 0.61 0.02 Gelbvieh 0.00 0.00 0.00 0.00 0.98 0.00 0.00 0.00 Gelbvieh0.01 0.00 0.01 0.00 0.92 0.01 0.04 0.00 Gelbvieh 0.03 0.00 0.01 0.000.94 0.01 0.01 0.01 Gelbvieh 0.00 0.00 0.00 0.00 0.98 0.00 0.00 0.00Gelbvieh 0.02 0.00 0.01 0.00 0.89 0.01 0.07 0.00 Gelbvieh 0.01 0.01 0.000.00 0.96 0.01 0.01 0.00 Gelbvieh 0.01 0.00 0.00 0.00 0.88 0.06 0.050.00 Hereford 0.01 0.95 0.01 0.00 0.01 0.01 0.01 0.01 Hereford 0.01 0.980.00 0.00 0.00 0.00 0.00 0.00 Hereford 0.01 0.97 0.00 0.00 0.01 0.000.00 0.00 Hereford 0.01 0.97 0.01 0.00 0.01 0.00 0.01 0.00 Hereford 0.010.93 0.02 0.00 0.01 0.00 0.01 0.04 Hereford 0.02 0.94 0.01 0.00 0.010.01 0.01 0.01 Hereford 0.01 0.97 0.00 0.00 0.01 0.00 0.01 0.00 Hereford0.00 0.98 0.00 0.00 0.00 0.00 0.00 0.00 Hereford 0.09 0.77 0.01 0.000.07 0.02 0.04 0.01 Hereford 0.01 0.98 0.00 0.00 0.01 0.00 0.00 0.00Hereford 0.00 0.98 0.00 0.00 0.00 0.00 0.00 0.00 Hereford 0.00 0.98 0.000.00 0.00 0.00 0.00 0.00 Hereford 0.01 0.97 0.00 0.00 0.01 0.00 0.000.00 Hereford 0.41 0.16 0.03 0.00 0.36 0.01 0.03 0.01 Hereford 0.00 0.990.00 0.00 0.00 0.00 0.00 0.00 Hereford 0.01 0.98 0.00 0.00 0.01 0.000.00 0.00 Hereford 0.12 0.74 0.01 0.00 0.08 0.02 0.03 0.01 Hereford 0.010.98 0.00 0.00 0.01 0.00 0.00 0.00 Hereford 0.01 0.97 0.00 0.00 0.010.00 0.00 0.00 Hereford 0.00 0.95 0.00 0.00 0.00 0.01 0.00 0.03 Hereford0.01 0.97 0.00 0.00 0.01 0.00 0.01 0.00 Hereford 0.00 0.99 0.00 0.000.00 0.00 0.00 0.00 Hereford 0.03 0.91 0.01 0.00 0.02 0.01 0.01 0.01Hereford 0.00 0.99 0.00 0.00 0.00 0.00 0.00 0.00 Hereford 0.01 0.96 0.010.00 0.01 0.01 0.01 0.00 Hereford 0.01 0.97 0.01 0.00 0.01 0.00 0.010.00 Limousin 0.01 0.01 0.01 0.00 0.01 0.97 0.01 0.00 Limousin 0.01 0.010.01 0.00 0.03 0.92 0.02 0.00 Limousin 0.01 0.00 0.01 0.00 0.01 0.970.01 0.00 Limousin 0.02 0.00 0.01 0.00 0.07 0.89 0.01 0.01 Limousin 0.010.00 0.00 0.00 0.01 0.97 0.01 0.00 Limousin 0.01 0.00 0.00 0.00 0.000.98 0.00 0.00 Limousin 0.01 0.00 0.00 0.00 0.01 0.95 0.02 0.00 Limousin0.01 0.01 0.00 0.00 0.01 0.96 0.01 0.00 Limousin 0.02 0.01 0.01 0.000.01 0.94 0.01 0.02 Limousin 0.01 0.00 0.00 0.00 0.01 0.97 0.01 0.00Limousin 0.02 0.02 0.02 0.00 0.05 0.86 0.04 0.01 Limousin 0.01 0.00 0.000.00 0.00 0.97 0.01 0.01 Limousin 0.01 0.00 0.00 0.00 0.01 0.97 0.010.00 Limousin 0.09 0.05 0.01 0.00 0.18 0.56 0.10 0.01 Limousin 0.29 0.010.01 0.00 0.01 0.67 0.01 0.00 Limousin 0.01 0.00 0.00 0.00 0.01 0.960.01 0.00 Limousin 0.00 0.00 0.00 0.00 0.01 0.97 0.01 0.01 Limousin 0.000.00 0.00 0.00 0.00 0.98 0.01 0.00 Limousin 0.14 0.00 0.02 0.01 0.010.80 0.03 0.00 Limousin 0.04 0.00 0.00 0.00 0.01 0.90 0.05 0.00 Limousin0.00 0.00 0.00 0.00 0.00 0.98 0.00 0.00 Limousin 0.01 0.00 0.00 0.000.01 0.97 0.01 0.00 Limousin 0.00 0.00 0.03 0.00 0.00 0.93 0.03 0.00Limousin 0.03 0.01 0.01 0.00 0.02 0.92 0.01 0.00 Limousin 0.01 0.01 0.090.00 0.01 0.69 0.18 0.00 Limousin 0.00 0.00 0.00 0.00 0.00 0.99 0.000.00 Simmental 0.35 0.02 0.01 0.00 0.15 0.01 0.45 0.01 Simmental 0.010.00 0.00 0.00 0.00 0.01 0.97 0.00 Simmental 0.01 0.00 0.00 0.00 0.010.00 0.96 0.01 Simmental 0.02 0.01 0.01 0.00 0.01 0.02 0.94 0.01Simmental 0.22 0.01 0.01 0.00 0.09 0.01 0.66 0.01 Simmental 0.01 0.000.00 0.00 0.01 0.01 0.97 0.00 Simmental 0.02 0.00 0.01 0.00 0.49 0.000.48 0.00 Simmental 0.01 0.00 0.00 0.00 0.01 0.01 0.96 0.01 Simmental0.02 0.01 0.00 0.00 G.02 0.01 0.90 0.04 Simmental 0.01 0.00 0.01 0.000.01 0.01 0.95 0.01 Simmental 0.02 0.00 0.01 0.01 0.01 0.01 0.95 0.00Simmental 0.00 0.00 0.00 0.00 0.01 0.00 0.98 0.00 Simmental 0.00 0.110.00 0.01 0.01 0.00 0.87 0.00 Simmental 0.02 0.01 0.01 0.00 0.02 0.370.57 0.01 Simmental 0.01 0.00 0.01 0.00 0.00 0.01 0.97 0.01 Simmental0.02 0.00 0.00 0.00 0.01 0.06 0.90 0.00 Simmental 0.01 0.00 0.03 0.000.02 0.01 0.93 0.00 Simmental 0.01 0.00 0.00 0.00 0.03 0.00 0.93 0.02Simmental 0.05 0.02 0.01 0.04 0.01 0.01 0.86 0.01 Simmental 0.03 0.010.03 0.00 0.02 0.01 0.91 0.00 Simmental 0.01 0.00 0.00 0.00 0.01 0.000.97 0.00 Simmental 0.01 0.00 0.00 0.00 0.01 0.01 0.96 0.00 Simmental0.02 0.00 0.00 0.00 0.10 0.38 0.48 0.00 Simmental 0.01 0.01 0.01 0.000.01 0.04 0.92 0.00

Example 3 Identification of Angus Vs Non-Angus

In another aspect of the invention, 12 SNP markers were selected andtested for their ability to distinguish Angus from non-Angus breeds.These markers included MMBT05243, MMBT02545, MMBT14829, MMBT11932,MMBT23373, MBT08423, MMBT19771, MMBT10324, MMBT01611, MMBT08985,MMBT02110, and MMBT17611 (See Tables 5 and 6). Parameters used in thisanalysis are shown in Table 9.

TABLE 9 Parameters used in the model Number of n (number B (number K(number Burn in Run SNP of of breeds of inferred period period markersanimals) in input) populations) (iterations) (iterations) 12 196 8 250,000 250,000 (Angus and non-Angus)

The mean individual admixture coefficient ({circumflex over (q)}) forAngus and non-Angus animals was 0.968 and 0.924, respectively,indicating a very high probability of assigning an animal from one ofthese populations to the correct cluster. The mean individual admixturecoefficient ({circumflex over (q)}) per animal is shown in Table 10.

TABLE 10 Mean individual admixture coefficient ({circumflex over (q)})per animal as an estimate of probability each individual animal belongsto a particular cluster. Inferred Breed of population animal 1 2 Angus0.99 0.02 Angus 1.00 0.00 Angus 0.98 0.02 Angus 0.99 0.01 Angus 1.000.01 Angus 0.99 0.01 Angus 0.99 0.01 Angus 0.40 0.60 Angus 0.98 0.02Angus 0.88 0.12 Angus 0.98 0.02 Angus 0.98 0.02 Angus 0.95 0.05 Angus0.99 0.01 Angus 0.99 0.01 Angus 0.97 0.03 Angus 0.95 0.05 Angus 0.990.01 Angus 0.46 0.54 Angus 0.95 0.05 Angus 0.97 0.03 Angus 0.98 0.02Angus 0.99 0.01 non Angus 0.02 0.98 non Angus 0.02 0.99 non Angus 0.020.98 non Angus 0.01 0.99 non Angus 0.02 0.98 non Angus 0.01 0.99 nonAngus 0.02 0.99 non Angus 0.01 0.99 non Angus 0.08 0.92 non Angus 0.310.69 non Angus 0.01 0.99 non Angus 0.02 0.98 non Angus 0.02 0.98 nonAngus 0.04 0.97 non Angus 0.01 0.99 non Angus 0.02 0.98 non Angus 0.040.96 non Angus 0.17 0.83 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.04 0.96 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.020.98 non Angus 0.04 0.96 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.58 0.42 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.01 1.00 non Angus 0.010.99 non Angus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.01 1.00 nonAngus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.011.00 non Angus 0.01 1.00 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.011.00 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 1.00 nonAngus 0.01 0.99 non Angus 0.33 0.67 non Angus 0.01 1.00 non Angus 0.011.00 non Angus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.34 0.66 non Angus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.010.99 non Angus 0.01 1.00 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 0.99 non Angus 0.05 0.95 non Angus 0.01 0.99 nonAngus 0.02 0.99 non Angus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.010.99 non Angus 0.02 0.98 non Angus 0.02 0.99 non Angus 0.01 0.99 nonAngus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.011.00 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.16 0.85 nonAngus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.01 0.99 nonAngus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.17 0.83 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.080.92 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 nonAngus 0.01 1.00 non Angus 0.07 0.93 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 1.00 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.011.00 non Angus 0.01 0.99 non Angus 0.40 0.60 non Angus 0.02 0.98 nonAngus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.26 0.74 non Angus 0.10 0.91 non Angus 0.02 0.98 nonAngus 0.01 0.99 non Angus 0.26 0.74 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.02 0.98 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.42 0.59 non Angus 0.01 0.99 non Angus 0.01 1.00 non Angus 0.011.00 non Angus 0.01 1.00 non Angus 0.01 1.00 non Angus 0.01 1.00 nonAngus 0.09 0.91 non Angus 0.01 0.99 non Angus 0.01 0.99 non Angus 0.010.99 non Angus 0.01 0.99 non Angus 0.28 0.73 non Angus 0.01 0.99 nonAngus 0.01 0.99 non Angus 0.10 0.90 non Angus 0.01 0.99 non Angus 0.050.95 non Angus 0.01 0.99 non Angus 0.02 0.98 non Angus 0.02 0.98 nonAngus 0.01 1.00 non Angus 0.01 0.99 non Angus 0.02 0.98 non Angus 0.010.99 non Angus 0.05 0.96 non Angus 0.03 0.98

This tool can be used to identify the percentage of each breedcomprising an individual animal. These data can be used to managefeedlot cattle for specific growth and development traits. In oneaspect, cattle identified from the exotic breed type (Charolais,Gelbvieh, Limousin, and Simmental) have high growth rate and largeharvest sizes. These cattle can be managed to maximize growth rate andlean meat yield. Cattle identified as English type (Angus and Hereford)produce high quality meat products and can be marketed into the highquality yield grade. Breed specified products can certify that theproduct is in fact from the breed labeled.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method to infer breed of a bovine subject froma nucleic acid sample of the bovine subject, comprising identifying inthe nucleic acid sample, at least one nucleotide occurrence of at leastone single nucleotide polymorphism (SNP) corresponding to position 300of any one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887 to 64895, whereinthe SNP is associated with a breed, thereby inferring the breed of thebovine subject.
 2. The method of claim 1, wherein the nucleotideoccurrence of at least 2 of the SNPs is determined to infer the breed.3. The method of claim 2, wherein the nucleotide occurrences of the atleast 2 SNPs comprise a haplotype allele, and wherein the method infersthe breed using the identified haplotype allele.
 4. The method of claim3, further comprising identifying a diploid pair of haplotype allelesand inferring the breed using the diploid pair of haplotype alleles. 5.The method of claim 1, wherein the identified nucleotide occurrence isassociated with Angus, Holstein, Limousin, Brahman, Simmental, Hereford,Gelbvieh or Charolais cattle.
 6. The method of claim 5, wherein at leastone nucleotide occurrence of at least one SNP listed in Table 5 or Table3A as associated with Angus cattle is identified.
 7. The method of claim5, wherein at least one nucleotide occurrence of at least one SNP listedin Table 5 or Table 3A as associated with Brahman cattle is identified.8. The method of claim 5, wherein at least one nucleotide occurrence ofat least one SNP listed in Table 5 or Table 3A as associated withLimousin cattle is identified.
 9. The method of claim 5, wherein atleast one nucleotide occurrence of at least one SNP listed in Table 5 orTable 3A as associated with Simmental cattle is identified.
 10. Themethod or claim 5, wherein at least one nucleotide occurrence of atleast one SNP listed in Table 5 or Table 3A as associated with Herefordcattle is identified.
 11. The method of claim 5, wherein at least onenucleotide occurrence of at least one SNP listed in Table 5 or Table 3Aas associated with Charolais cattle is identified.
 12. The method ofclaim 5, wherein at least one nucleotide occurrence of at least one SNPlisted in Table 5 or Table 3A as associated with Gelbvieh cattle isidentified.
 13. A method for sorting bovine subjects, comprising: a.inferring a breed for a first bovine subject from a nucleic acid sampleof the first bovine subject, by a method comprising identifying anucleotide occurrence of at least one single nucleotide polymorphism(SNP) corresponding to position 300 of at least one of SEQ ID NOS:1 to4868 or SEQ ID NOS:64887 to 64895, wherein the SNP is associated withthe breed; and b. sorting the first bovine subject based on the inferredbreed, and repeating for additional subjects, thereby sorting bovinesubjects.
 14. The method of claim 13, wherein the bovine subjects aresorted based on whether they are Angus, Limousin, Brahman, Simmental,Hereford, Gelbvieh or Charolais cattle.
 15. A method for breeding abovine subject, comprising: a. inferring breed of a bovine candidate foruse in breeding programs from a nucleic acid sample of the bovinecandidate by a method comprising identifying the nucleotide occurrenceof at least one single nucleotide polymorphism (SNP) corresponding toposition 300 of at least one of SEQ ID NOS:1 to 4868 or SEQ ID NOS:64887to 64895, wherein the SNP is associated with breed; and b. determiningwhether to select the individual for use in breeding programs based onthe inferred breed, thereby breeding the bovine subject.
 16. The methodof claim 15, wherein the selection is implemented at the elite orbreeding nucleus level or at the multiplier or foundation animal level.17. The method of claim 15, wherein the selected bovine subject is usedfor breeding.
 18. A bovine subject resulting from the selection andbreeding according to claim
 15. 19. The method of claim 18, wherein theselected bovine subject is used for cloning.